JP2018524334A - Thienopyrrole compounds and their use as inhibitors of Oplophorus-derived luciferase - Google Patents

Abstract

Disclosed are thienopyrrole compounds capable of inhibiting Oplophorus-derived luciferase, as well as compositions and kits comprising the thienopyrrole compounds, and methods of using the thienopyrrole compounds. 【Selection chart】 None

Description

This application claims the benefit of US Provisional Patent Application No. 62 / 184,714 filed Jun. 25, 2015, and US Provisional Patent Application No. No. 206,525, the entire contents of which are incorporated herein by reference.

  The present disclosure relates to thienopyrrole compounds capable of inhibiting Oplophorus-derived luciferase.

  Reporter molecules are routinely used to monitor molecular events in the fields of biology, biochemistry, immunology, cell biology and molecular biology. Luciferases based on luciferases secreted from deep-sea shrimp Shrimp (Oplophorus gracilirostris) may be used as a reporter molecule and has advantageous features including broad substrate specificity, high activity, and high quantum yield It is shown to have In certain applications it may be further advantageous to control the luminescence signal from Oplophorus luciferase.

In one aspect, the disclosure provides a compound of formula (I), or a salt thereof:
(I)
During the ceremony:
The dotted lines indicate the presence or absence of a bond;
n is 0, 1, 2, 3, 4 or 5;
X is CH, N, O, or S;
When the dotted line represents the presence of a bond, X is CH or N, and when the dotted line represents the absence of a bond, X is O or S;
A is an optionally substituted phenyl ring or an optionally substituted 5 or 6 membered heteroaryl ring;
R ¹ and R ² are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally And R ³ and R ⁴ are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally selected from the group consisting of: alkoxyalkyl substituted with and optionally substituted alkoxyalkoxyalkyl; And C ₃ -C ₈ -cycloalkyl, optionally substituted C ₃ -C ₈ -cycloalkylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl , Optionally selected from the group consisting of optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, and optionally substituted heterocyclylalkyl Or R ³ and R ⁴ together with the nitrogen atom to which they are attached form an optionally substituted ring.

  In one aspect, the disclosure relates to contacting an Oplophorus-derived luciferase with a compound described herein, such as a compound of Formula (I), (Ia), (Ib), (Ib '), or (II). Provided are methods of inhibiting Oplophorus-derived luciferase, including:

In one aspect, the disclosure provides a method of inhibiting an Oplophorus-derived luciferase, comprising contacting the Oplophorus-derived luciferase with a compound of Formula (II):
(II)
During the ceremony:
The dotted lines indicate the presence or absence of a bond;
n is 0, 1, 2, 3, 4 or 5;
X is CH, N, O, or S;
When the dotted line represents the presence of a bond, X is CH or N, and when the dotted line represents the absence of a bond, X is O or S;
A is an optionally substituted phenyl ring or an optionally substituted 5 or 6 membered heteroaryl ring;
R ¹ and R ² are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally Selected from the group consisting of alkoxyalkyl substituted with and optionally substituted alkoxyalkoxyalkyl;
Z is selected from the group consisting of -NR ³ R ⁴ and -OR ⁵ ; and R ³ , R ⁴ and R ⁵ are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, Optionally substituted C ₃ -C ₈ -cycloalkyl, optionally substituted C ₃ -C ₈ -cycloalkylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted hetero Selected from the group consisting of aryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, and optionally substituted heterocyclylalkyl; or R ³ and R ⁴ with the nitrogen atom to which they are attached Together, form an optionally substituted ring together.

In one aspect, the disclosure is a method of modulating the luminescence of an Oplophorus-derived luciferase in a sample,
(A) contacting the sample with a coelenterazine substrate and a compound as described herein, such as a compound of formula (I), (Ia), (Ib), (Ib ') or (II); and (b) The method provides a method that comprises detecting luminescence in a sample, wherein the compound causes a decrease in luminescence from an Oplophorus-derived luciferase.

In one aspect, the disclosure is a method of detecting an interaction between a first protein and a second protein in a sample, the method comprising:
(A) contacting the sample with a coelenterazine substrate and a compound as described herein, such as a compound of Formula (I), (Ia), (Ib), (Ib '), or (II);
(I) a first polynucleotide encoding a first fusion protein comprising a first fragment of Oplophorus-derived luciferase and a first protein; and (ii) a second fragment and a second protein of Oplophorus-derived luciferase A second polynucleotide encoding a second fusion protein; and (b) detecting luminescence in the sample,
Provided is a method wherein the detection of luminescence is indicative of the interaction between the first protein and the second protein.

In one aspect, the disclosure is a method of detecting an interaction between a first protein and a second protein in a sample, the method comprising:
(A) contacting the sample with a coelenterazine substrate and a compound as described herein, such as a compound of Formula (I), (Ia), (Ib), (Ib '), or (II);
(Iii) a first polynucleotide encoding a first fusion protein comprising a bioluminescent donor Oplophorus-derived luciferase and a first protein; and (iv) a second fusion comprising a fluorescent acceptor molecule and a second protein A second polynucleotide encoding a protein;
Including
(B) detecting a bioluminescence resonance energy transfer (BRET) in a sample indicative of the interaction or proximity of the bioluminescence donor and the fluorescence acceptor.

  In one aspect, the disclosure provides a first fusion protein comprising a first target protein and a bioluminescent donor molecule, wherein the bioluminescent donor molecule is an Oplophorus-derived luciferase; and a second target protein and a fluorescent acceptor molecule. Bioluminescence comprising a second fusion protein comprising: a coelenterazine substrate and a compound as described herein, such as a compound of formula (I), (Ia), (Ib), (Ib ') or (II) Provided is a resonant energy transfer (BRET) system.

In one aspect, the disclosure is
A kit comprising: (a) a compound as described herein, such as a compound of formula (I), (Ia), (Ib), (Ib ') or (II); and (b) an Oplophorus-derived luciferase Do.

In one aspect, the disclosure is a method of modulating the luminescence of an Oplophorus-derived luciferase in a sample,
(A) contacting a sample with coelenterazine substrate and a compound of formula (II),
(II)
During the ceremony:
The dotted lines indicate the presence or absence of a bond;
n is 0, 1, 2, 3, 4 or 5;
X is CH, N, O, or S;
When the dotted line represents the presence of a bond, X is CH or N, and when the dotted line represents the absence of a bond, X is O or S;
A is an optionally substituted phenyl ring or an optionally substituted 5 or 6 membered heteroaryl ring;
R ¹ and R ² are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally Selected from the group consisting of alkoxyalkyl substituted with and optionally substituted alkoxyalkoxyalkyl;
Z is selected from the group consisting of -NR ³ R ⁴ and -OR ⁵ ; and R ³ , R ⁴ and R ⁵ are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, Optionally substituted C ₃ -C ₈ -cycloalkyl, optionally substituted C ₃ -C ₈ -cycloalkylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted hetero Selected from the group consisting of aryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, and optionally substituted heterocyclylalkyl; or R ³ and R ⁴ with the nitrogen atom to which they are attached Together, forming together optionally substituted rings, contacting,
(B) detecting luminescence in the sample;
Compounds of formula (II) provide a method that causes a decrease in luminescence from an Oplophorus-derived luciferase.

In one aspect, the disclosure is a method of detecting an interaction between a first protein and a second protein in a sample, the method comprising:
(A) contacting a sample with coelenterazine substrate and a compound of formula (II),
(II)
During the ceremony:
The dotted lines indicate the presence or absence of a bond;
n is 0, 1, 2, 3, 4 or 5;
X is CH, N, O, or S;
When the dotted line represents the presence of a bond, X is CH or N, and when the dotted line represents the absence of a bond, X is O or S;
A is an optionally substituted phenyl ring or an optionally substituted 5 or 6 membered heteroaryl ring;
R ¹ and R ² are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally Selected from the group consisting of alkoxyalkyl substituted with and optionally substituted alkoxyalkoxyalkyl;
Z is selected from the group consisting of -NR ³ R ⁴ and -OR ⁵ ; and R ³ , R ⁴ and R ⁵ are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, Optionally substituted C ₃ -C ₈ -cycloalkyl, optionally substituted C ₃ -C ₈ -cycloalkylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted hetero Selected from the group consisting of aryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, and optionally substituted heterocyclylalkyl; or R ³ and R ⁴ with the nitrogen atom to which they are attached Together, form an optionally substituted ring together,
The sample is
(V) a first polynucleotide encoding a first fusion protein comprising a first fragment of Oplophorus-derived luciferase and a first protein; and (vi) a second fragment and a second protein of Oplophorus-derived luciferase Contacting comprising a second polynucleotide encoding a second fusion protein; and (b) detecting luminescence in the sample,
Detection of luminescence provides a method that indicates the interaction between the first protein and the second protein.

In one aspect, the disclosure is a method of detecting an interaction between a first protein and a second protein in a sample, the method comprising:
(A) contacting a sample with coelenterazine substrate and a compound of formula (II),
(II)
During the ceremony:
The dotted lines indicate the presence or absence of a bond;
n is 0, 1, 2, 3, 4 or 5;
X is CH, N, O, or S;
When the dotted line represents the presence of a bond, X is CH or N, and when the dotted line represents the absence of a bond, X is O or S;
A is an optionally substituted phenyl ring or an optionally substituted 5 or 6 membered heteroaryl ring;
R ¹ and R ² are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally Selected from the group consisting of alkoxyalkyl substituted with and optionally substituted alkoxyalkoxyalkyl;
Z is selected from the group consisting of -NR ³ R ⁴ and -OR ⁵ ; and R ³ , R ⁴ and R ⁵ are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, Optionally substituted C ₃ -C ₈ -cycloalkyl, optionally substituted C ₃ -C ₈ -cycloalkylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted hetero Selected from the group consisting of aryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, and optionally substituted heterocyclylalkyl; or R ³ and R ⁴ with the nitrogen atom to which they are attached Together, form an optionally substituted ring together,
The sample is
(Vii) a first polynucleotide encoding a first fusion protein comprising a bioluminescent donor Oplophorus-derived luciferase and a first protein; and (viii) a second fusion comprising a fluorescent acceptor molecule and a second protein A second polynucleotide encoding a protein;
Contacting, including;
(B) Providing a method comprising detecting bioluminescence resonance energy transfer (BRET) in a sample indicative of the interaction or proximity of a bioluminescence donor and a fluorescence acceptor.

In one aspect, the disclosure provides a first fusion protein comprising a first target protein and a bioluminescent donor molecule, wherein the bioluminescent donor molecule is an Oplophorus-derived luciferase; and a second target protein and a fluorescent acceptor molecule. Providing a bioluminescent resonance energy transfer (BRET) system comprising a second fusion protein comprising: coelenterazine substrate, and a compound of formula (II),
(II)
During the ceremony:
The dotted lines indicate the presence or absence of a bond;
n is 0, 1, 2, 3, 4 or 5;
X is CH, N, O, or S;
When X represents CH or N when the dotted line represents the presence of a bond, X is O or S when the dotted line represents the absence of a bond;
A is an optionally substituted phenyl ring, or an optionally substituted 5 or 6 membered heteroaryl ring;
R ¹ and R ² are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally Selected from the group consisting of alkoxyalkyl substituted with and optionally substituted alkoxyalkoxyalkyl;
Z is selected from the group consisting of -NR ³ R ⁴ and -OR ⁵ ; and R ³ , R ⁴ and R ⁵ are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, Optionally substituted C ₃ -C ₈ -cycloalkyl, optionally substituted C ₃ -C ₈ -cycloalkylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted hetero Selected from the group consisting of aryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, and optionally substituted heterocyclylalkyl; or R ³ and R ⁴ with the nitrogen atom to which they are attached Together, form an optionally substituted ring together.

In one aspect, the disclosure is
(A) Compound of Formula (II):
(II)
During the ceremony:
The dotted lines indicate the presence or absence of a bond;
n is 0, 1, 2, 3, 4 or 5;
X is CH, N, O, or S;
When the dotted line represents the presence of a bond, X is CH or N, and when the dotted line represents the absence of a bond, X is O or S;
A is an optionally substituted phenyl ring or an optionally substituted 5 or 6 membered heteroaryl ring;
R ¹ and R ² are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally Selected from the group consisting of alkoxyalkyl substituted with and optionally substituted alkoxyalkoxyalkyl;
Z is selected from the group consisting of -NR ³ R ⁴ and -OR ⁵ ; and R ³ , R ⁴ and R ⁵ are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, Optionally substituted C ₃ -C ₈ -cycloalkyl, optionally substituted C ₃ -C ₈ -cycloalkylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted hetero Selected from the group consisting of aryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, and optionally substituted heterocyclylalkyl; or R ³ and R ⁴ with the nitrogen atom to which they are attached Together, they form an optionally substituted ring together; and (b) a kit comprising an Oplophorus-derived luciferase.

16 shows inhibition of the NANOLUC® (Nluc) enzyme by JRW-0004 in a multiplex assay combining RealTime-Glo and the CASPASE-GLO® assay system. Figure 7 shows the inhibition of Nluc by the thienopyrrole compounds JRW-0004, JRW-0013, JRW-0006, JRW-0042, JRW-0138, and JRW-0147. It is shown that the thienopyrrole compounds JRW-0004, JRW-0013, JRW-0006, JRW-0042, JRW-0138, and JRW-0147 do not inhibit firefly luciferase activity. FIG. 4 shows that the thienopyrrole compounds JRW-0004 (FIGS. 4A and 4B) and WZ141-88 (FIGS. 4C and 4D) inhibit Nluc in a dose and time dependent manner. The thienopyrrole compounds WZ141-86 (FIGS. 5A and 5B), WZ141-74 (FIG. 5C), and WZ141-84 (FIG. 5D) inhibit Nluc in a dose- and time-dependent manner. FIG. 6 shows that the thienopyrrole compounds WZ141-89 (FIG. 6A), WZ141-90 (FIG. 6B), and WZ141-91 (FIG. 6C) inhibit Nluc in a dose- and time-dependent manner. Thienopyrrole compounds JRW-0138 (FIG. 7A), JRW-0140 (FIG. 7B), JRW-0142 (FIG. 7C), JRW using HEK293 or HeLa cells transiently transfected with β2 adrenergic receptor-Nluc fusion protein The permeabilities of -0143 (Figure 7D), JRW-0145 (Figure 7E), and JRW-0147 (Figure 7F) are shown. Thienopyrrole compounds JRW-0148 (FIG. 8A), JRW-0149 (FIG. 8B), JRW-0151 (FIG. 8C), JRW using HEK293 or HeLa cells transiently transfected with β2 adrenergic receptor-Nluc fusion protein The permeability of -0152 (Figure 8D), JRW-0051 (Figure 8E), and JRW-0043 (Figure 8F) is shown. Thienopyrrole compounds JRW-0044 (FIG. 9A), JRW-0013 (FIG. 9B), JRW-0034 (FIG. 9C), JRW using HEK293 or HeLa cells transiently transfected with β2 adrenergic receptor-Nluc fusion protein The permeabilities of -0052 (Figure 9D), JRW-0110 (Figure 9E), and JRW-0187 (Figure 9F) are shown. Thienopyrrole compounds JRW-0188 (Figure 10A), JRW-0190 (Figure 10B), JRW-0191 (Figure 10C), JRW using HEK 293 or HeLa cells transiently transfected with β2 adrenergic receptor-Nluc fusion protein. The permeability of -0192 (Figure 10D), JRW-0195 (Figure 10E), and JRW-0197 (Figure 10F) is shown. The thienopyrrole compounds JRW-0198 (FIG. 11A), JRW-0200 (FIG. 11B), and JRW-0208 (FIG. 11C) using HEK293 or HeLa cells transiently transfected with a β2 adrenergic receptor-Nluc fusion protein It shows permeability. Figure 7 shows the ability of thienopyrrole compounds to inhibit extracellular BRET. FIG. 12A shows a schematic of simulated extracellular BRET assay design. 12B-12E show compounds of thienopyrrole compounds JRW-0013 (FIG. 12B), JRW-0051 (FIG. 12C), JRW-0147 (FIG. 12D), and JRW-0187 (FIG. 12E) using an extracellular BRET assay. The reaction curve is shown. The ability of thienopyrrole compounds to inhibit extracellular luciferase activity and to increase intracellular BRET is shown. FIG. 13A shows a schematic of mock extracellular Nluc assay design. 13B-13E show compounds of the thienopyrrole compounds JRW-0013 (FIG. 13B), JRW-0051 (FIG. 13C), JRW-0147 (FIG. 13D), and JRW-0187 (FIG. 13E) using an extracellular Nluc assay. The reaction curve is shown. Figure 20 shows cell permeability of thienopyrrole compounds JRW-0147, JRW-0051, and JRW-0138. Figure 20 shows cell impermeability of the thienopyrrole compound JRW-0147 in a target association model. FIG. 15A shows a schematic of SRC-Nluc assay design. 15B-13C show the compound response curves of dasatinib-DY 607 (FIG. 15B) and JRW-0147 (FIG. 15C). The permeability of the thienopyrrole compounds JRW-0147 and JRW-0013 is shown. Permeability of the thienopyrrole compounds JRW-0013 (FIG. 17A), JRW-0147 (FIG. 17B), and JRW-0344 (FIG. 17C) using HEK293 cells transiently transfected with the β2 adrenergic receptor-Nluc fusion protein Indicates FIG. 18 shows cell viability and toxicity of JRW-0344 (FIG. 18A) compared to digitonin (FIG. 18B) and DMSO (FIG. 18C). Figure 20 shows cell permeability of thienopyrrole compounds JRW-0147, JRW-0344, and JRW-0013. Figure 19 shows the ability of the thienopyrrole compound JRW-0344 to inhibit extracellular luciferase activity. FIG. 20A shows a schematic of the SRC-Nluc assay design. 20B and 20C show compound reaction curves of the thienopyrrole compound JRW-0344 using the SRC-Nluc assay (FIG. 20B) or the extracellular Nluc assay (FIG. 20C).

  Also disclosed herein are Oplophorus-derived luciferases, eg, the luciferase of SEQ ID NO: 2 (also referred to herein interchangeably as "NanoLuc", "Nluc", "Nluc luciferase", and "Nluc enzyme" A thienopyrrole compound capable of selectively inhibiting Due to the stability and potential of being excreted from cells, it may be advantageous to use a selective inhibitor which suppresses the light emission from the Oplophorus derived luciferase in certain applications. For example, in applications involving time multiplexing of multiple luminescence systems, it may be beneficial to have selective inhibitors per system so that only one luminescence signal can be measured and / or detected at a time. Furthermore, in some plate-based assays, some amount of luciferase can be excreted from the cells. Extracellular inhibitor compounds can help to improve the signal to noise ratio in certain assays, as they can selectively suppress luminescence from excreted luciferase.

  Thienopyrrole compounds described herein have been discovered to be selective inhibitors of Oplophorus luciferase. Thienopyrrole compounds can compete with the binding of the coelenterazine substrate of luciferase and can be modified to produce both cell permeable and cell impermeable inhibitors.

1. Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Although methods and materials similar or equivalent to those described herein can be used in the practice and testing of the present invention, preferred methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are hereby incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

  The terms "comprise (s)", "include (s)", "having", "has", "can", "contain (s)" and their variants as used herein are additional It is intended to be an unconstrained conventional phrase, term or word which does not exclude the possibility of an act or structure. The singular forms "a", "and" and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that “comprise”, “consistent of”, and “consisting essentially of” the embodiments and elements presented herein, whether or not explicitly referred to. Do.

  As used herein, the terms "substituent" or "suitable substituent" refer to a chemically acceptable functional group, such as a moiety that does not abolish the activity of the compound of the present invention. Intended. Illustrative examples of suitable substituents include halo, perfluoroalkyl, perfluoroalkoxy, alkyl, alkenyl, alkynyl, hydroxy, halo, oxo, mercapto, alkylthio, alkoxy Group, nitro group, azide alkyl group, sulfonic acid group, aryl or heteroaryl group, aryloxy or heteroaryloxy group, aralkyl or heteroaralkyl group, aralkoxy or heteroaralkoxy group, HO- (C = O)-group , Heterocyclic group, cycloalkyl group, amino group, alkyl- and dialkylamino group, carbamoyl group, alkylcarbonyl group, alkylcarbonyloxy group, alkoxycarbonyl group, alkylaminocarbonyl group, dialkylaminocarbonyl group, arylcarbonyl group , An aryloxycarbonyl group, an alkylsulfonyl group, and a arylsulfonyl group include, but are not limited to. The substituents may be substituted by additional substituents. The substituent may be in the form of a salt (for example, the sulfonic acid group may be in the form of a sulfonate group).

  As used herein, the term "alkenyl" contains 2 to 10 carbons and is linear or branched containing at least one carbon-carbon double bond formed by the removal of two hydrogens. Chain refers to a hydrocarbon chain. Representative examples of alkenyl include ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl and 3-decenyl. Examples include, but are not limited to. The alkenyl groups of the present invention may be unsubstituted or substituted with one or more suitable substituents, preferably 1 to 3, suitable substituents as defined above.

  As used herein, the term "alkoxy" refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy and hexyloxy.

  As used herein, the term "alkoxyalkoxy" is an alkoxy as defined herein appended to the parent molecular moiety through another alkoxy group as defined herein. Point to a group. Representative examples of alkoxyalkoxy include, but are not limited to, tert-butoxymethoxy, 2-ethoxyethoxy, 2-methoxyethoxy, and methoxymethoxy.

  As used herein, the term "alkoxyalkoxyalkyl" is an alkoxyalkoxy as defined herein appended to the parent molecular moiety through an alkylene group as defined herein. Means a group. Representative examples of alkoxyalkoxyalkyl include, but are not limited to, tert-butoxymethoxymethyl, ethoxymethoxymethyl, (2-methoxyethoxy) methyl, and 2- (2-methoxyethoxy) ethyl.

  As used herein, the term "alkoxyalkyl" refers to an alkoxy group as defined herein appended to the parent molecular moiety through an alkyl group as defined herein. Point to. Representative examples of alkoxyalkyl include, but are not limited to, tert-butoxymethyl, 2-ethoxyethyl, 2-methoxyethyl, and methoxymethyl.

  As used herein, the term "alkoxycarbonyl" refers to an alkoxy group as defined herein appended to the parent molecular moiety through a carbonyl group as defined herein. Point to. Representative examples of alkoxycarbonyl include, but are not limited to, methoxycarbonyl, ethoxycarbonyl and tert-butoxycarbonyl.

  As used herein, the term "alkoxycarbonylalkyl" is an alkoxycarbonyl as defined herein appended to the parent molecular moiety through an alkylene group as defined herein. Means a group. Representative examples of alkoxycarbonylalkyl include, but are not limited to, ethoxycarbonylmethyl, 3-methoxycarbonylpropyl, 4-ethoxycarbonylbutyl, and 2-tert-butoxycarbonylethyl.

As used herein, the term "alkyl" preferably has 1 to 30 carbon atoms, 1 to 12 carbon atoms, 1 to 10 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbons It refers to a straight or branched hydrocarbon radical having an atom, or 1 to 4 carbon atoms. The term "C ₁ -C ₈ alkyl" is defined to include alkyl groups having carbon atoms of 7 or 8 in the arrangement of straight or branched chain. For example, “C ₁ -C ₈ alkyl” specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl (eg, n-pentyl), hexyl (E.g. n-hexyl), heptyl (e.g. n-heptyl) and octyl (e.g. n-octyl). The term "C ₁ -C ₆ alkyl" is defined to include alkyl groups having carbon atoms of 2, 3, 4, 5 or 6 in the arrangement of straight or branched chain. For example, specific examples of “C ₁ -C ₆ alkyl” include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl (eg, n-pentyl), and And hexyl (e.g. n-hexyl). The term "C ₁ -C ₄ - alkyl" is defined to include alkyl groups having carbon atoms of 1, 2, 3 or 4, in the arrangement of straight or branched chain. For example, “C ₁ -C ₄ -alkyl” specifically includes methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, isobutyl and tert-butyl. The alkyl groups of the present invention may be unsubstituted or substituted with one or more suitable substituents, preferably 1 to 3 suitable substituents as defined above. For example, an alkyl group may be substituted with one or more halo substituents to form a haloalkyl group, or may be substituted with one or more hydroxy substituents to form a hydroxyalkyl group, Alternatively, it may be substituted with one or more alkoxy groups to form an alkoxyalkyl group.

  As used herein, the term "alkylamino" refers to an alkyl group as defined herein appended to the parent molecular moiety through an amino group as defined herein. Point to. Representative examples of alkylamino include, but are not limited to, methylamino, ethylamino, isopropylamino, butyl-amino and sec-butylamino.

  As used herein, the term "alkylaminoalkyl" is an alkyl as defined herein appended to the parent molecular moiety through an aminoalkyl group as defined herein. Point to a group. Representative examples of alkylaminoalkyl groups include, but are not limited to, methylaminoethyl and methylamino-2-propyl.

  As used herein, the term "alkylcarbonyl" refers to an alkyl group as defined herein appended to the parent molecular moiety through a carbonyl group as defined herein. Point to. Representative examples of alkylcarbonyl include, but are not limited to, acetyl, 1-oxopropyl, 2,2-dimethyl-1-oxopropyl, 1-oxobutyl, and 1-oxopentyl.

  As used herein, the term "alkylcarbonylalkyl" is an alkylcarbonyl as defined herein appended to the parent molecular moiety through an alkyl group as defined herein. Point to a group.

  As used herein, the term "alkylcarbonylalkylamide" is attached to the parent molecular moiety through an alkylamide group as defined herein, as defined herein. It refers to an alkylcarbonyl group.

The term "alkylene" refers to a saturated, linear or branched hydrocarbon derived divalent group of 1 to 10 carbon atoms. Representative examples of _{alkylene, -CH 2 -, - CH (} CH 3) -, - C (CH 3) 2 -, - CH 2 CH 2 -, - CH 2 CH 2 CH 2 -, - CH 2 CH 2 _{CH 2 CH 2 -, - CH} 2 C (CH 3) 2 -, and _{-CH 2 CH (CH 3) CH} 2 - include, but are not limited to.

  As used herein, the term "alkynyl" has 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbons and has one or more carbon-carbon triple bonds It refers to a linear or branched hydrocarbon radical. Alkynyl groups of the present invention include, but are not limited to ethynyl, propynyl and butynyl. The alkynyl groups of the present invention may be unsubstituted or substituted with one or more suitable substituents, preferably 1 to 3, suitable substituents as defined above.

As used herein, the term "amido", a carbonyl group, as defined herein (ie, -CONH ₂₎ is appended to the parent molecular moiety through a refers to an amino group. As used herein, the term "alkylamide" refers to an alkylamino or dialkylamino group appended to the parent molecular moiety through a carbonyl group as defined herein. Representative examples of alkylamides include, but are not limited to, methylaminocarbonyl, dimethylaminocarbonyl, ethylmethylaminocarbonyl, and n-hexylaminocarbonyl.

As used herein, the term "amino" refers to the group -NH _2.

  As used herein, the term "aminoalkyl" is attached to the parent molecular moiety through an alkyl group as defined herein, at least one as defined herein. It refers to an amino group. Representative examples of aminoalkyl include, but are not limited to, aminomethyl, 2-aminoethyl, 3-aminopropyl, 4-aminobutyl, 5-aminopentyl, and 6-aminohexyl.

  As used herein, the term "aminoalkylamide" is at least one defined herein, appended to the parent molecular moiety through an alkylamide group as defined herein. Refers to such an amino group.

As used herein, the term "amino protecting group" refers to a moiety that prevents a chemical reaction from occurring on the nitrogen atom to which the protecting group is attached. Also, the amino protecting group should be removable by chemical reaction. Such groups are well known in the art and are described in Protecting Groups in Organic Synthesis, T., et al. W. Greene and P. G. M. Wuts, 3 ^rd edition, contains John Wiley & Sons, 1999 to those as specifically described, in its entirety by reference to the be incorporated in this document. Suitable amino protecting groups, carbobenzyloxy _{(-NHCO-OCH 2 C 6 H} 5 or -NH-Cbz); t- butyloxycarbonyl _{(-NHCO-OC (CH 3)} 3 or -NH-Boc); 9-fluorenylmethyloxycarbonyl (-NH-Fmoc), 2,2,2-trichloroethyloxycarbonyl (-NH-Troc), and allyloxycarbonyl (-NH-Alloc) include but is not limited to I will not. (In each of the above, -NH- represents nitrogen from a protected amino group.)

  As used herein, the term "aminoluciferin" refers to (4S) -2- (6-amino-1,3-benzothiazol-2-yl) -4,5-dihydrothiazole-4-carboxylic acid Or a substituted analog of this molecule.

  As used herein, the term "aryl" means a monocyclic, bicyclic or tricyclic aromatic radical. Representative examples of aryl groups include, but are not limited to, phenyl, dihydroindenyl, indenyl, naphthyl, dihydronaphthalenyl and tetrahydronaphthalenyl. The aryl groups of the present invention may be optionally substituted by one or more suitable substituents, preferably 1 to 5 suitable substituents as defined above.

  As used herein, the term "arylalkyl" refers to an aryl group as defined herein appended to the parent molecular moiety through an alkyl group as defined herein. Point to. Representative examples of arylalkyl include, but are not limited to, phenylmethyl (ie, benzyl) and phenylethyl.

  As used herein, the term "arylcarbonyl" refers to an aryl group as defined herein appended to the parent molecular moiety through a carbonyl group as defined herein. Point to.

  As used herein, the terms "bioluminescence" or "luminescence" may refer to light produced as a result of a reaction between an enzyme and a substrate that produces light. Examples of such enzymes (bioluminescent enzymes) include Oplophorus luciferases such as Oplophorous gracilirostris, firefly luciferases such as Photinus pyralis or Photuris pennsylvanica, click beetle luciferase, Renilla luciferase, sea firefly luciferase, aequorin photoprotein, oberin photoprotein, etc. Can be mentioned.

  As used herein, the terms "carbonyl" or "(C = O)" (as used in terms such as alkylcarbonyl, alkyl- (C = O)-or alkoxycarbonyl) are alkyl Refers to the attachment of a> C = O moiety to a second moiety such as a group or an amino group (ie, an amide group). Alkoxycarbonylamino (i.e., alkoxy (C = O) -NH-) refers to an alkyl carbamate group. A carbonyl group is also defined equally as (C = O) herein. Alkylcarbonylamino refers to groups such as acetamide.

  As used herein, the term "carboxy" refers to a -C (O) OH group.

  As used herein, the term "carboxyalkyl" refers to a carboxy group as defined herein appended to the parent molecular moiety through an alkyl group as defined herein. Point to.

  As used herein, the term "carboxyalkylamide" is a carboxyalkyl as defined herein appended to the parent molecular moiety through an amide group as defined herein. Point to a group.

  As used herein, the term "coelenterazine substrate" refers to a class of reporter molecules that emit light when acted upon by various bioluminescent proteins such as luciferase (eg, marine luciferase). Coelenterazine substrates include coelenterazine and its analogs and derivatives.

  As used herein, the term "cycloalkyl" is a monocyclic, bicyclic or tricyclic carbocyclic radical optionally containing one or two double bonds (eg, cyclopropyl, cyclobutyl , Cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclopentenyl, cyclohexenyl, bicyclo [2.2.1] heptanyl, bicyclo [3.2.1] octanyl and bicyclo [5.2.0] nonanyl etc.) Point to The cycloalkyl groups of the present invention may be unsubstituted or substituted with one or more suitable substituents, preferably 1 to 5 suitable substituents as defined above.

  As used herein, the term "cycloalkylalkyl" is a cycloalkyl as defined herein appended to the parent molecular moiety through an alkyl group as defined herein. Point to a group. Representative examples of cycloalkylalkyl include, but are not limited to, cyclohexylmethyl.

  As used herein, the term "cycloalkylamide" is a cycloalkyl as defined herein appended to the parent molecular moiety through an amide group as defined herein. Point to a group.

  As used herein, the term "dialkylamino" is two, as defined herein, appended to the parent molecular moiety through an amino group, as defined herein. It refers to an independently selected alkyl group. Representative examples of dialkylamino include, but are not limited to, N, N-dimethylamino, N-ethyl-N-methylamino, and N-isopropyl-N-methylamino.

  As used herein, the term "dialkylaminoalkyl" is a dialkylamino as defined herein appended to the parent molecular moiety through an alkyl group as defined herein. Point to a group. Representative examples of dialkylaminoalkyl include, but are not limited to, N, N-dimethylaminoethyl and N, N-methyl (2-propyl) aminoethyl.

  As used herein, the term "dialkylaminoalkylamide" is attached to the parent molecular moiety through an alkylamide group as defined herein, as defined herein. It refers to a dialkylamino group.

  As used herein, the term "halogen" or "halo" refers to a fluoro, chloro, bromo or iodo radical.

  As used herein, the term "haloalkoxy" refers to an alkoxy group as defined herein substituted by 1, 2, 3 or 4 halogen atoms. Representative examples of haloalkoxy include, but are not limited to, chloromethoxy, 2-fluoroethoxy, trifluoromethoxy and pentafluoroethoxy.

  As used herein, the term "haloalkyl" refers to an alkyl group as defined herein substituted by 1, 2, 3 or 4 halogen atoms. Representative examples of haloalkyl include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, 2-chloro-3-fluoropentyl and 4,4,4-trifluorobutyl .

  As used herein, the term "heteroaryl" refers to monocyclic heteroaryl or bicyclic heteroaryl. Monocyclic heteroaryl is a 5- or 6-membered ring. The 5-membered ring contains two double bonds. The 5-membered ring may contain one heteroatom selected from O or S; or one, two, three, four or nitrogen atoms and optionally one oxygen or sulfur atom. The 6-membered ring contains 3 double bonds and 1, 2, 3 or 4 nitrogen atoms. Representative examples of monocyclic heteroaryl include furanyl, imidazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, 1,3-oxazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyrrolyl, tetrazolyl, thiadiazolyl, 1,3-thiazolyl, Examples include, but are not limited to thienyl, triazolyl and triazinyl. As bicyclic heteroaryl, monocyclic heteroaryl fused to phenyl, monocyclic heteroaryl fused to monocyclic cycloalkyl, or monocyclic heteroaryl fused to monocyclic cycloalkenyl Or monocyclic heteroaryl fused to monocyclic heteroaryl, or monocyclic heteroaryl fused to monocyclic heterocycle. Representative examples of bicyclic heteroaryl include benzofuranyl, benzothienyl, benzoxazolyl, benzimidazolyl, benzooxadiazolyl, 6,7-dihydro-l, 3-benzothiazolyl, imidazo [l, 2-a] pyridinyl Indazolyl, indolyl, isoindolyl, isoquinolinyl, naphthyridinyl, pyridoimidazolyl, quinazolinyl, quinolinyl, thiazolo [5,4-b] pyridin-2-yl, thiazolo [5,4-d] pyrimidin-2-yl and 5,6 , 7,8-tetrahydroquinolin-5-yl, but is not limited thereto. The heteroaryl groups of the present invention may be unsubstituted or substituted with one or more suitable substituents, preferably 1 to 5 suitable substituents as defined above.

  As used herein, the term "heteroarylalkyl" is a heteroaryl as defined herein appended to the parent molecular moiety through an alkyl group as defined herein. Point to a group. Representative examples of heteroarylalkyl include flu-3-ylmethyl, 1H-imidazol-2-ylmethyl, 1H-imidazol-4-ylmethyl, 1- (pyridin-4-yl) ethyl, pyridin-3-ylmethyl, 6- Chloropyridin-3-ylmethyl, pyridin-4-ylmethyl, (6- (trifluoromethyl) pyridin-3-yl) methyl, (6- (cyano) pyridin-3-yl) methyl, (2- (cyano) pyridine -4-yl) methyl, (5- (cyano) pyridin-2-yl) methyl, (2- (chloro) pyridin-4-yl) methyl, pyrimidin-5-ylmethyl, 2- (pyrimidin-2-yl) Examples include, but are not limited to, propyl, thien-2-ylmethyl, and thien-3-ylmethyl.

As used herein, the term "heterocycle" or "heterocyclyl" refers to a monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle. Monocyclic heterocycles are 3, 4, 5, 6, 7 or 8 membered rings containing at least one heteroatom independently selected from the group consisting of oxygen, nitrogen, phosphorus and sulfur. The 3- or 4-membered ring contains zero or one double bond and one heteroatom selected from the group consisting of oxygen, nitrogen, phosphorus and sulfur. The 5-membered ring contains zero or one double bond and one, two or three heteroatoms selected from the group consisting of oxygen, nitrogen, phosphorus and sulfur. The 6-membered ring contains zero, one or two double bonds and one, two or three heteroatoms selected from the group consisting of oxygen, nitrogen, phosphorus and sulfur. The 7- and 8-membered rings contain zero, one, two or three double bonds and one, two or three heteroatoms selected from the group consisting of oxygen, nitrogen, phosphorus and sulfur. Representative examples of monocyclic heterocyclic rings include azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, and the like. Isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, phosphinan, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl , Tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidine Le, thiomorpholinyl, 1,1-di-oxide thiomorpholinyl (thiomorpholine sulfone), thiopyranyl, trithianyl and 2,5-dioxo - pyrrolidinyl include, but are not limited to. The bicyclic heterocycle is a monocyclic heterocycle fused to a phenyl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl, or a monocyclic fused to a monocyclic cycloalkenyl A monocyclic heterocycle fused to a heterocycle of the formula or a monocyclic heterocycle, or an alkylene bridge or 2, 3 or 4 carbon atoms in which two non-adjacent atoms of the ring are 1, 2, 3 or 4 Ring systems of bridged monocyclic heterocycles linked by alkenylene bridges of 4 carbon atoms. Representative examples of the bicyclic heterocyclic ring include benzopyranyl, benzothiopyranyl, chromanyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothienyl, azabicyclo [2.2.1] heptyl (2- Azabicyclo [2.2.1] hept-2-yl), 2,3-dihydro-lH-indolyl, isoindolinyl, octahydrocyclopenta [c] pyrrolyl, octahydropyrrolopyridinyl, 9-phosphabicyclo Examples include, but are not limited to, [3.3.1] nonane, 8-phosphabicyclo [3.2.1] octane, and tetrahydroisoquinolinyl. The tricyclic heterocycle is a bicyclic heterocycle fused to a phenyl group, or a bicyclic heterocycle fused to a monocyclic cycloalkyl, or a bicyclic fused to a monocyclic cycloalkenyl A heterocycle of the formula or a bicyclic heterocycle fused to a monocyclic heterocycle, or an alkylene bridge of the two non-adjacent atoms of the bicyclic ring of 1, 2, 3 or 4 carbon atoms or It is exemplified by a bicyclic heterocycle linked by an alkenylene bridge of 2, 3 or 4 carbon atoms. Examples of tricyclic heterocycles include octahydro-2,5-epoxypentalene, hexahydro-2H-2,5-methanocyclopenta [b] furan, hexahydro-lH-1,4-methanocyclopenta [c Furan, aza-admantane (1-azatricyclo [3.3.1.1 ^3,7 ] decane), oxa-adamantane (2-oxatricyclo [3.3.1.1 ^3,7 ] decane) and 2 , 4,6-trioxa-8-phosphatricyclo [3.3.1.13,7] decane, but is not limited thereto. The heterocyclic groups of the present invention may be unsubstituted or substituted with one or more suitable substituents, preferably 1 to 3 suitable substituents as defined above. The heterocyclic groups of the present invention may contain one or more oxo (= O) or thioxo (= S) groups attached to the ring.

  As used herein, the term "heterocyclylalkyl" refers to a heterocyclyl group as defined herein appended to the parent molecular moiety through an alkyl group as defined herein. Point to. Representative examples of heterocyclylalkyl include piperidin-4-ylmethyl, piperazin-1-ylmethyl, 3-methyl-1-pyrrolidin-1-ylbutyl, (1R) -3-methyl-1-pyrrolidin-1-ylbutyl, (1S Examples include, but are not limited to) -3-methyl-1-pyrrolidin-1-ylbutyl and 3-morpholinopropyl.

  As used herein, the term "heterocyclylamido" refers to a heterocyclyl group as defined herein appended to the parent molecular moiety through an amido group as defined herein. Point to.

  As used herein, the term "hydroxy" refers to an -OH group.

  As used herein, the term "hydroxyalkoxy" refers to an alkoxy group as defined herein substituted by at least one hydroxy group. Representative examples of hydroxyalkoxy include, but are not limited to, hydroxyethoxy and 2-hydroxypropoxy.

  As used herein, the term "hydroxyalkyl" refers to an alkyl group as defined herein substituted by at least one hydroxy group. Representative examples of hydroxyalkyl include hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, 2,3-dihydroxypropyl, 2,3-dihydroxypentyl, 4-hydroxybutyl, 2-ethyl-4-hydroxyheptyl, 3 Include, but are not limited to, 4-dihydroxybutyl and 5-hydroxypentyl.

  As used herein, the term "hydroxyalkylamide" refers to a hydroxyalkyl group attached to an amido group, such as -amido-alkyl-OH.

  As used herein, the term "hydroxycarbonyl" refers to a hydroxy group as defined herein appended to the parent molecular moiety through a carbonyl group as defined herein .

  The terms "luminescent enzyme", "bioluminescent enzyme" or "luciferase", as used interchangeably herein, refer to the class of oxidizing enzymes used in bioluminescence, wherein the enzyme receives light when given a substrate. Generate and emit light. The luciferase may be a naturally occurring, recombinant or mutant luciferase using a luciferase substrate. The luciferase substrate may be luciferin, a luciferin derivative or analog, a preluciferin derivative or analog, coelenterazine, or coelenterazine derivative or analog. The luminescent enzyme, if naturally occurring, may be easily obtained from the organism by one skilled in the art. If the luminescent enzyme is a naturally occurring or recombinant one or a mutant luminescent enzyme, for example, one that retains the activity of the naturally occurring luminescent enzyme in the luciferase-coelenterazine reaction or the luciferase-luciferin reaction If any, it can be easily obtained from cultures of bacteria, yeast, mammalian cells, insect cells, plant cells, etc. transformed to express nucleic acid encoding the luminescent enzyme. Furthermore, recombinant or mutant luminescent enzymes can be derived from in vitro cell release systems using nucleic acid encoding luciferase. Suitable luminescent enzymes include Oplophoidea (eg luciferase from Oplophorus), beetle luciferases (eg Photinus pyralis, Photuris pennsylvanica etc.), cnidarians (eg Renilla luciferase), Aristeidae, Solenoceridae, Luciferidae, Sergestidae, Pasipheidae and Luciferases from bioluminescent decapods, such as from marine organisms such as the Thalassocarididae decapod family, Gaussia luciferases such as Gaussia princeps luciferase, Metridia longa luciferase and Metridia pacifica Luci Included are Metridia luciferases such as ferrase, Vargula luciferases such as Vargula hilgendorfii luciferase, copepods luciferases such as Pleuromamma xiphias luciferase, and photoproteins such as Aequorin, as well as mutants, recombinants and mutants thereof.

  A "luminescent reaction mixture" contains substances that will allow the luminescent enzyme to generate a light signal, ie luminescence. The mixture may also contain an enzyme, such as a luciferase enzyme or luciferase. The substances and particular concentrations and / or amounts required to generate the luminescent signal will vary depending on the luminescent enzyme used and the type of assay performed. Buffer to maintain the reaction at an appropriate pH, eg, other additives including additives such as PRIONEX or bovine serum albumin (BSA), reducing agents, surfactants etc to help maintain enzyme activity Substances will often be added to the solution.

As used herein, the term "methylenedioxy" refers to -OCH ₂ O-group in which an oxygen atom in the methylenedioxy are attached to the parent molecular moiety through two adjacent carbon atoms.

  As used herein, the terms "Oplophorus luciferase" and "Oplophorus-derived luciferase" are used interchangeably and include deep-sea shrimp Oplophorus gracilirostris, including its wild-type, mutants, and mutants (eg, Refers to luciferase secreted from SEQ ID NO: 1). For example, suitable Oplophorus luciferase mutants are described in US Pat. Nos. 8,557,970 and 8,669,103, the entire contents of each of which are incorporated herein by reference. Do. Exemplary Oplophorus-derived luciferases include, for example, those of SEQ ID NO: 2 (also referred to herein interchangeably as "NanoLuc", "Nluc", "Nluc luciferase", and "Nluc enzyme").

  As used herein, the term "oxo" refers to a doubly bonded oxygen (= O) radical whose binding partner is a carbon atom. Such radicals can be thought of as carbonyl groups.

  The terms "peptide" or "polypeptide" refer to a sequence of at least two amino acids. In some embodiments, the peptide may contain 80 or fewer amino acids, or 35 or fewer amino acids, or 10 or fewer amino acids or 5 or fewer amino acids.

  As used herein, the term "reporter moiety" can mean a moiety that directly or indirectly generates a detectable signal under suitable conditions. Exemplary reporter moieties include, but are not limited to, fluorophores, luminescent molecules, dyes, radioactive labels, and substrates for enzymes such as luciferase. In some embodiments, for example, where the reporter moiety is a substrate for an enzyme, the reporter moiety may indirectly generate a detectable signal. A reaction between the substrate and the enzyme takes place, producing a detectable signal such as fluorescence or luminescence. As used herein, the term "bioluminescent reporter moiety" may refer to a moiety that is a substrate for luciferase. For example, the bioluminescent reporter moiety may be a luciferin, luciferin precursor, aminoluciferin, quinolyl-luciferin, naphthylluciferin, fluoroluciferin, luciferin derivatives such as fluoroluciferin, chloroluciferin, precursors of luciferin derivatives, coelenterazine, or coelenterazines such as coelenterazine derivatives or flimazine It may be analog. The luminescence signal generated may be detected using a luminescence meter. As used herein, the term "fluorescent reporter moiety" may mean a moiety that fluoresces. For example, the fluorescent reporter moiety may be a fluorophore such as coumarin, R110, fluorescein, DDAO, resorufin, cresyl violet, silyl xanthene, or carbopyronine. The fluorescence may be detected using a fluorometer.

The prefix given to a multicomponent substituent only applies to the first component that precedes it. For example, the term "cycloalkylalkyl" contains two components: alkyl and cycloalkyl. Thus, a C ₁ -C ₆ prefix in C ₁ -C ₆ cycloalkylalkyl means that the alkyl component of the cycloalkylalkyl contains 1 to 6 carbon atoms; the C ₁ -C ₆ prefix is: The cycloalkyl component is not described. By way of further example, the term "halo-C ₁ -C ₆ -alkyl" refers to halomethyl, haloethyl, halopropyl, halobutyl, halopentyl and halohexyl. By way of further example, the prefix "halo" in haloalkoxyalkyl indicates that only the alkoxy component of the alkoxyalkyl substituent is substituted with one or more halogen radicals. If halogen substitution only occurs at the alkyl component, the substituent will instead be described as "alkoxyhaloalkyl".

  A substituent is "substitutable" if it contains at least one carbon atom or nitrogen atom attached to one or more hydrogen atoms. Thus, for example, hydrogen, halogen and cyano do not fall within the scope of this definition. In addition, the sulfur atom in heterocyclyl containing such atoms can be substituted with one or two oxo substituents.

  If a substituent is described as being "substituted", the non-hydrogen radical is in the position of a hydrogen radical on carbon or nitrogen of the substituent. Thus, for example, a substituted alkyl substituent is an alkyl substituent in which at least one non-hydrogen radical is in the position of an alkyl substituent hydrogen radical. For example, monofluoroalkyl is alkyl substituted with a fluoro radical, and difluoroalkyl is alkyl substituted with two fluoro radicals. It should be appreciated that each non-hydrogen radical may be the same or different (unless otherwise stated), provided there is more than one substitution on the substituents.

  When a substituent is said to be "unsubstituted" or not "substituted" or "optionally substituted" it is meant that the substituent does not have any substituent. If a substituent is described as "optionally substituted," the substituent is either (1) unsubstituted or (2) substituted. If a substituent is described as optionally substituted by up to a specified number of non-hydrogen radicals, then that substituent is (1) not substituted or (2) a specified number of non-hydrogen radicals Up to, or up to the maximum number of substitutable positions on the substituent may be substituted, whichever is less. Thus, for example, if the substituent is described as heteroaryl optionally substituted by up to three non-hydrogen radicals, then heteroaryl having less than three substitutable positions is that heteroaryl Is optionally substituted by up to the same number of non-hydrogen radicals as having a substitutable position. For example, tetrazolyl (with only one substitutable position) will be optionally substituted by up to one non-hydrogen radical. For example, if it is stated that the amino nitrogen is optionally substituted by up to 2 non-hydrogen radicals, then the primary amino nitrogen is optionally substituted by up to 2 non-hydrogen radicals While, the secondary amino nitrogen will be optionally substituted by at most one non-hydrogen radical.

  If a substituent is described as being “independently selected” from groups, each substituent is selected independently of the other. Thus, each substituent may be the same as or different from the other substituents.

  In the description of several ranges herein, each number intervening therebetween having the same degree of accuracy is expressly contemplated. For example, for the range 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, for the range 6.0 to 7.0 the numbers 6.0, 6.1, 6.2. 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

2. Thienopyrrole Compounds Provided herein are thienopyrrole compounds capable of inhibiting Oplophorus-derived luciferase and / or Oplophorus-derived luciferase activity. Thienopyrrole compounds include compounds of formula (I) and salts thereof:
(I)
During the ceremony:
The dotted lines indicate the presence or absence of a bond;
n is 0, 1, 2, 3, 4 or 5;
X is CH, N, O, or S;
When the dotted line represents the presence of a bond, X is CH or N, and when the dotted line represents the absence of a bond, X is O or S;
A is an optionally substituted phenyl ring or an optionally substituted 5 or 6 membered heteroaryl ring;
R ¹ and R ² are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally And R ³ and R ⁴ are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally selected from the group consisting of: alkoxyalkyl substituted with and optionally substituted alkoxyalkoxyalkyl; And C ₃ -C ₈ -cycloalkyl, optionally substituted C ₃ -C ₈ -cycloalkylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl , Optionally selected from the group consisting of optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, and optionally substituted heterocyclylalkyl Or R ³ and R ⁴ together with the nitrogen atom to which they are attached form an optionally substituted ring.

  In some embodiments, the dotted line represents the presence of a bond and X is CH.

  In some embodiments, n is one.

  In some embodiments, A is a phenyl ring. In some embodiments, A is a 5-membered heteroaryl ring. In some embodiments, A is a thienyl ring. In some embodiments, A is a furanyl ring.

In some embodiments, R ¹ is selected from the group consisting of hydrogen, C ₁ -C ₈ alkyl, halo-C ₁ -C ₈ -alkyl, alkoxyalkoxyalkyl, and arylalkyl. For example, in some embodiments, R ¹ is selected from the group consisting of hydrogen, ethyl, n-hexyl, 2- (2-methoxyethoxy) ethyl and benzyl. In some embodiments, R ¹ is ethyl.

In some embodiments, R ² is optionally substituted aryl. For example, in some embodiments, R ² is substituted phenyl. R ² is phenyl substituted with one substituent selected from the group consisting of C ₁ -C ₄ alkyl, cyano, amido, C ₁ -C ₄ alkoxy, and hydroxyalkyl. In some embodiments, R ² is phenyl substituted with one methyl group (eg, m-tolyl).

In some embodiments, R ¹ is C ₁ -C ₄ alkyl and R ² is optionally substituted phenyl. In some embodiments, R ¹ is ethyl and R ² is m-tolyl.

In some embodiments, R ³ and R ⁴ together with the nitrogen atom to which they are attached form an optionally substituted ring together. In some embodiments, R ³ and R ⁴ together with the nitrogen atom to which they are attached form an optionally substituted monocyclic heterocycle. In some embodiments, the optionally substituted monocyclic heterocycle is selected from the group consisting of optionally substituted pyrrolidine, piperidine and piperazine. In some embodiments, the optionally substituted monocyclic heterocycle is unsubstituted pyrrolidine, unsubstituted piperidine, piperidine substituted with one substituent (eg, alkoxycarbonyl such as ethoxycarbonyl), or 1 One of a substituent piperazines (eg, C ₁ -C ₄ alkyl such as methyl) is selected from the group consisting of.

In some embodiments, R ³ is hydrogen.

In some embodiments, R ⁴ is unsubstituted C ₁ -C ₈ alkyl (eg, methyl), halo-C ₁ -C ₈ -alkyl, carboxy-C ₁ -C ₈ -alkyl (eg,-(CH 2) _{2) 7 -COOH), C 1} -C 4 - alkoxycarbonyl -C ₁ -C ₈ - alkyl _{(e.g., - (CH 2) 7 -COOCH} 3), phenyl (hereinafter which is optionally substituted), an optionally substituted has been C ₅ -C ₆ cycloalkyl (e.g., unsubstituted cyclopentyl, unsubstituted cyclohexyl or substituted cyclohexyl), optionally substituted heterocyclyl (for example, unsubstituted piperidinyl, tert- butoxycarbonyl, -CO- (CH ₂₎ alkoxycarbonyl alkylcarbonyl such as ₄ -COOCH _3, or -CO- (CH _{2) 4} -COOH in carboxyalkylcarbonyl such Conversion piperidinyl) heteroarylalkyl, which are optionally substituted (e.g., -CH ₂ - pyridyl -C ₁ -C _4, such as pyridyl - alkyl), and optionally substituted heterocyclylalkyl (for example, - (CH ₂ And the like) selected from the group consisting of morpholino-C ₁ -C ₄ -alkyl) such as ₃ -morpholino. For example, in some embodiments, R ⁴ is unsubstituted or C ₁ -C ₄ -alkoxycarbonyl (eg, -C (O) OCH ₃ ) and C ₁ -C ₄ -alkoxycarbonyl-C ₁ -C ₄ - alkyl _{(e.g., -CH 2 -C (O) CH} 2 CH 3) phenyl substituted with one substituent such as substituents selected from the group consisting of.

In some embodiments, R ⁴ is carboxy, C ₁ -C ₄ -alkoxycarbonyl (eg, -C (O) OCH ₃ ), C ₁ -C ₈ -alkylamide (eg, -C (O) NH 2 -(CH ₂ ) ₃ CH ₃ or -C (O) NH- (CH ₂ ) ₅ CH ₃ ), hydroxy-C ₁ -C ₈ -alkylamide (eg, -C (O) NH- (CH ₂ ) ₂ OH or _{-C (O) NH- (CH 2} ) 6 OH), amide (ie, -CONH _2), amino -C substituted with optionally ₁ -C ₈ - alkyl amide (e.g., -C (O) NH -(CH ₂ ) ₆ NH ₂ ), C ₁ -C ₄ -dialkylamino-C ₁ -C ₈ -alkylamide (eg -C (O) NH- (CH ₂ ) ₂ N (CH ₃ ) ₂ ), Carboxy-C ₁ -C ₈ -alkyl amides (eg, -C (O) NH- (CH ₂ ) ₃ CO OH, -C (O) NH- ( CH 2) 5 COOH or _{-C (O) NH- (CH 2} ) 7 COOH), sulfonic acid -C ₁ -C ₈ - alkyl amide (e.g., -C (O) _{NH- (CH 2) 6 SO 3} H), sulfonic acid -C ₁ -C ₈ - alkyl amide (e.g., -C (O) NH- (CH 2) 6 SO 3 -), C 1 -C 4 - alkyl Carbonyl-C ₁ -C ₈ -alkyl amides (eg, —C (O) NH— (CH ₂ ) ₃ COOCH ₃ , —C (O) NH— (CH ₂ ) ₅ COOCH ₃ or —C (O) NH— (CH ₂ ) ₇ COOCH ₃ ), optionally substituted C ₃ -C ₆ -cycloalkyl amides such as, for example, -C (O) NH-cyclohexyl, -C (O) NH-cyclohexyl-COOH or -C (O ) NH- cyclohexyl -COOCH _3), and Heteroshikurirua De (e.g., -C (O) NH- piperidinyl, unsubstituted or methyl substituted with tert- butoxycarbonyl or acetyl) cyclohexyl substituted with one substituent selected from the group consisting of. For example, in some embodiments, R ⁴ is optionally substituted amino-C ₁ -C ₈ -alkyl amide (eg, —C (O) NH— (CH ₂ ) ₆ NH ₂ ), amino The group is protected with an amino protecting group (e.g. tert-butoxycarbonyl) or the amino group is protonated to form a salt (e.g. a hydrochloride salt) or the amino group is a fluorophore (e.g. fluorescein) Or polypeptide (eg -Asp-Asp-Asp peptide, optionally acetylated at the end).

In some embodiments, the compound has Formula (Ia):
(Ia)
Wherein R ³ and R ⁴ are as defined in any of the embodiments described above for formula (I).

In some embodiments, the compound has Formula (Ib):
(Ib)
During the ceremony:
Y is selected from the group consisting of -NR ^a R ^b and -OR ^c ;
R ^a and R ^b are each independently of the group consisting of hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally substituted C ₃ -C ₈ -cycloalkyl, and optionally substituted heterocyclyl Or R ^a and R ^b together with the nitrogen atom to which they are attached together form an optionally substituted ring; and R ^c is hydrogen and optionally substituted C ₁ It is selected from the group consisting of -C ₄ alkyl.

In some embodiments, Y is -OR ^c . In some embodiments, R ^c is selected from the group consisting of hydrogen and methyl.

In some embodiments, Y is -NR ^a R ^b.

In some embodiments, R ^a is hydrogen. In some embodiments, R ^b is selected from the group consisting of hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally substituted C ₃ -C ₈ -cycloalkyl, and optionally substituted heterocyclyl It is selected. In some embodiments, R ^b is hydrogen, unsubstituted C ₁ -C ₆ alkyl (eg, n-butyl or n-hexyl), hydroxyalkyl (eg, (CH ₂ ) ₂ OH or-(CH ₂ ) Hydroxy-C ₁ -C ₆ -alkyl such as ₆ OH, optionally substituted aminoalkyl (eg amino-C ₁ -C ₆ -alkyl such as-(CH ₂ ) ₆ NH ₂ ), dialkylaminoalkyl (eg For example, C ₁ -C ₄ -dialkylamino-C ₁ -C ₈ -alkylamides such as -C (O) NH- (CH ₂ ) ₂ N (CH ₃ ) ₂ , carboxyalkyl (eg-(CH ₂₎ )) ₃ COOH,-(CH ₂ ) ₅ COOH or-(CH ₂ ) ₇ COOH and like carboxy -C ₁ -C ₈ -alkyl), sulfonic acid -C ₁ -C ₈ -alkyl (eg-(CH ₂ ) ₆ SO _{3 H),} sul Phosphate -C ₁ -C ₈ - alkyl _{(e.g., - (CH 2) 6 SO} 3 -), alkylcarbonylalkyl _{(e.g., - (CH 2) 3 COOCH} 3, - (CH 2) 5 COOCH 3 or - ( C ₁ -C ₄ -alkylcarbonyl-C ₁ -C ₈ -alkylamide such as CH ₂ ) ₇ COOCH ₃ , optionally substituted C ₃ -C ₆ -cycloalkyl (eg -cyclohexyl, -cyclohexyl-COOH or - cyclohexyl -COOCH _3), and 6-membered heterocyclyl optionally substituted (e.g., unsubstituted or methyl, is selected from the group consisting of substituted) with tert- butoxycarbonyl or acetyl. For example, in some embodiments, R ^b is optionally substituted amino-C ₁ -C ₈ -alkyl (eg,-(CH ₂ ) ₆ NH ₂ ) and the amino group is an amino protecting group ( For example, protected with tert-butoxycarbonyl) or the amino group is protonated to form a salt (eg, a hydrochloride salt), or the amino group can be a fluorophore (eg, fluorescein) or a polypeptide (eg, It is further functionalized with -Asp-Asp-Asp peptide, optionally terminally acetylated).

In some embodiments, R ^a and R ^b together with the nitrogen atom to which they are attached form an optionally substituted ring together. In some embodiments, R ^a and R ^b together with the nitrogen atom to which they are attached form an optionally substituted monocyclic heterocycle. In some embodiments, the optionally substituted monocyclic heterocycle is an optionally substituted piperidine. In some embodiments, the monocyclic heterocycle is selected from the group consisting of unsubstituted piperidine and piperidine substituted with one substituent (eg, a carboxyl such as -C (O) OCH ₂ CH ₃ Or alkoxycarbonyl).

In some embodiments, the compound has the following Formula (Ib ′):
(Ib ')
Wherein Y is as defined in any of the embodiments described above for Formula (Ib).

  Suitable compounds include the following:

  N-Cyclohexyl-4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;

  N-ethyl-2- (5- (pyrrolidine-1-carbonyl) -4H-thieno [3,2-b] pyrrol-4-yl) -N- (m-tolyl) acetamide;

  N-ethyl-2- (5- (piperidine-1-carbonyl) -4H-thieno [3,2-b] pyrrol-4-yl) -N- (m-tolyl) acetamide;

  1- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carbonyl) piperidine-4-carboxylate;

  4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N-phenyl-4H-thieno [3,2-b] pyrrole-5-carboxamide;

  2- (4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) phenyl) ethyl acetate;

  Methyl 3- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) benzoate;

  Methyl-cis-4- (4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid;

  8- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) octanoic acid;

  6- (4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) piperidin-1-yl) -6 -Oxohexanoic acid;

  Trans-methyl-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid;

  Trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid;

  N- (trans-4- (butylcarbamoyl) cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;

  4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (trans-4-((2-hydroxyethyl) carbamoyl) cyclohexyl) -4H-thieno [3,2-b] pyrrole -5- carboxamide;

  N- (trans-4-((2- (dimethylamino) ethyl) carbamoyl) cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2- b] pyrrole-5-carboxamide;

  4- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1-carboxamide) Butanoic acid;

  N- (trans-4-carbamoylcyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;

  4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (trans-4- (hexylcarbamoyl) cyclohexyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;

  1- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carbonyl) Ethyl piperidine-4-carboxylate;

  6- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxamide) Methyl hexanoate;

  6- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxamide) Hexanoic acid;

  1- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carbonyl) Piperidine-4-carboxylic acid;

  8- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxamide) Octanoic acid;

  N- (trans-4- (cyclohexylcarbamoyl) cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;

  4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (trans-4-((1-methylpiperidin-4-yl) carbamoyl) cyclohexyl) -4H-thieno [3,2 -B] pyrrole-5-carboxamide;

  4- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1-carboxamide) Piperidine-1-carboxylic acid tert-butyl;

  4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (trans-4- (piperidin-4-ylcarbamoyl) cyclohexyl) -4H-thieno [3,2-b] pyrrole- 5-carboxamide;

  N- (trans-4-((1-acetylpiperidin-4-yl) carbamoyl) cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2 -B] pyrrole-5-carboxamide;

  (6- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1-carboxamide ) Hexyl) tert-butyl carbamate;

  N- (trans-4-((6- (3 ′, 6′-dihydroxy-3-oxo-3H-spiro [isobenzofuran-1,9′-xanthene] -5 (6) -carboxamido) hexyl) carbamoyl) Cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;

  N- (trans-4-((6-aminohexyl) carbamoyl) cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole -5-carboxamide hydrochloride;

  4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (trans-4-((6-hydroxyhexyl) carbamoyl) cyclohexyl) -4H-thieno [3,2-b] pyrrole -5- carboxamide;

  Methyl-trans-4- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane 1-carboxamide) cyclohexane-1-carboxylic acid;

  Trans-4- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1- Carboxamide) cyclohexane-1-carboxylic acid;

  (11S, 14S, 17S) -17-acetamido-11, 14-bis (carboxymethyl) -1- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl)- 4 H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexyl) -1,10,13,16-tetraoxo-2,9,12,15-tetraazanonadecane-19-oic acid;

  4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N-methyl-4H-thieno [3,2-b] pyrrole-5-carboxamide;

  N-Cyclopentyl-4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;

  4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (pyridin-4-ylmethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;

  4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (3-morpholinopropyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;

  N-ethyl-2- (5- (4-methylpiperazine-1-carbonyl) -4H-thieno [3,2-b] pyrrol-4-yl) -N- (m-tolyl) acetamide;

  Methyl 4- (2-oxo-2- (m-tolylamino) ethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate;

  N-Cyclohexyl-4- (2-oxo-2- (m-tolylamino) ethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;

  N-Cyclohexyl-4- (2- (2- (2- (2-methoxyethoxy) ethyl) (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;

  Methyl-trans-4- (4- (2- (2- (2-methoxyethoxy) ethyl) (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5 -Carboxamide) cyclohexane 1-carboxylic acid;

  4- (2- (Hexyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate methyl;

  N-cyclohexyl-4- (2- (hexyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;

  Methyl-trans-4- (4- (2- (hexyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid;

  4- (2- (benzyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate methyl;

  6- (cis-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxamide) Hexanoic acid;

  6- (trans-4-((4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) methyl) cyclohexane-1 -Carboxamide) methyl hexanoic acid;

  6- (trans-4-((4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) methyl) cyclohexane-1 -Carboxamide) hexanoic acid;

  6- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxamide) Sodium hexane-1-sulfonate;

  6- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxamide) Hexane-1-sulfonic acid potassium;

  Trans-4- (4- (2- (Hexyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1-carboxylic acid;

  Trans-4- (4- (2- (ethyl (phenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid methyl;

  Trans-4- (4- (2-((3-Cyanophenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid Methyl;

  Trans-4- (4- (2-((3-carbamoylphenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid Methyl;

  Trans-4- (4- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid methyl;

  Trans-4- (4- (2- (ethyl (3-methoxyphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid methyl;

  Trans-4- (4- (2- (ethyl (o-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid methyl;

  Trans-4- (4- (2- (6-methyl-3,4-dihydroquinolin-1 (2H) -yl) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide ) Methyl cyclohexane-1 -carboxylate;

  Trans-4- (4- (2- (ethyl (p-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid methyl;

  Trans-4- (4- (2- (ethyl (4- (hydroxymethyl) phenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carvone Methyl acid;

  Trans-4- (1- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -1H-indole-2-carboxamide) methyl cyclohexane-1-carboxylate;

  Trans-4- (1- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -6-methoxy-1H-indole-2-carboxamido) cyclohexane-1-carboxylate methyl;

  Trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-furo [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid methyl;

  4- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -N- (trans-4-((6-hydroxyhexyl) carbamoyl) cyclohexyl) -4H-thieno [3,2-b] Pyrrole-5-carboxamide;

  6- (trans-4- (4- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1-carboxamide ) Hexane-1-sulfonic acid sodium;

  Trans-4- (4- (2- (ethyl (3-isopropylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid methyl;

  Trans-4- (4- (2- (ethyl (3- (hydroxymethyl) phenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid Methyl acid;

  Trans-4- (4- (2-((3- (bromomethyl) phenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1- Methyl carboxylate;

  Trans-4- (4- (2-((3- (dimethylamino) phenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1 -Methyl carboxylate;

  Trans-4- (4- (2- (ethyl (3-isobutylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid methyl;

  Trans-4- (1- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -1H-indole-2-carboxamide) methyl cyclohexane-1-carboxylate;

  1- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -N- (trans-4-((6-hydroxyhexyl) carbamoyl) cyclohexyl) -1H-indole-2-carboxamide;

  6- (trans-4- (1- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -1H-indole-2-carboxamido) cyclohexane-1-carboxamido) hexane-1-sulfonic acid sodium ;

  Trans-4- (1- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -1H-pyrrole-2-carboxamide) methyl cyclohexane-1-carboxylate;

  Methyl trans-4- (6- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -6H-thieno [2,3-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylate.

(1) Salt Form The thienopyrrole compound described herein may be in the form of a salt. The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salt is, for the purposes of this disclosure, the parent form of the compound. Is equivalent to

For example, if the compound has a functional group that may be anionic or may be anionic (e.g., -COOH may be -COO-), a suitable cation may be used to form a salt. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na ⁺ and K ⁺ , alkaline earth cations such as Ca2 ⁺ and Mg2 ⁺ , and other cations. Examples of suitable organic cations include ammonium ion (ie NH ₄ ⁺ ), and substituted ammonium ion (eg NH ₃ R ₁ ⁺ , NH ₂ R ₂ ⁺ , NHR ₃ ⁺ , NR ₄ ⁺ ) and the like However, it is not limited to these. Examples of some suitable substituted ammonium ions are those derived from ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine and tromethamine And amino acids such as lysine and arginine.

Compound may be either or cationic cationic functional groups (e.g., -NH ₂ may may be -NH ₃ ^+), then a can to form a salt with a suitable anion. Examples of suitable inorganic anions include those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid However, it is not limited to these.

  Examples of suitable organic anions include the following organic acids: 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, edetic acid, ethanedisulfonic acid, ethane Sulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalene carboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucus acid, Derived from oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric acids But there are But it is not limited to these. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid and carboxymethylcellulose.

  Unless otherwise stated, references to the particular thienopyrrole compounds herein also include their salt forms.

(2) Isomer One particular thienopyrrole compound is one or more specific, geometric isomers, optical isomers, enantiomers, diastereomers, epimers, atropes, stereoisomers, tautomers, configurations. Geoisomers, or anomer forms, such as, but not limited to, cis and trans; E and Z; c, t and r; endo and exo; R and S And meso form; D form and L form; d form and l form; (+) form and (-) form; keto form, enol form and enolate form; syn form and anti form; syncrinal form and anti clinal form; There are, but not limited to, β form; axial form and equatorial form; wedge form, chair form, twist form, envelope form and half chair form; and combinations thereof. In the following, they are collectively referred to as "isomers" (or "isomeric forms").

  In some embodiments, the compounds described herein may be enantiomerically enriched isomers of the stereoisomers described herein. For example, the compound is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80 It may have an enantiomeric excess of%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. As used herein, enantiomer refers to any of a pair of chemical compounds whose molecular structures are mirror images of one another.

  In some embodiments, preparations of compounds disclosed herein are enriched in isomers of compounds having selected stereochemistry, eg, R or S, corresponding to the selected stereocenter. For example, the compound has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the selected stereocenters It has a purity corresponding to the compound having the selected stereochemistry.

  In some embodiments, the compositions described herein are enriched in a structure or structures having a selected stereochemistry, eg, R or S at a selected stereocenter Included are preparations of the disclosed compounds. Exemplary R / S configurations can be provided in the examples described herein.

  An "enriched preparation" as used herein is rich in selected configurations of one, two, three or more selected stereocenters within the compound of interest. Exemplary selected stereocenters, and exemplary configurations thereof, can be selected, for example, from those provided herein in the examples described herein. Enriched means, for example, that the compound molecule having the selected stereochemistry of the selected stereocenter is at least 60% in the preparation. In certain embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%. Abundance refers to the level of the molecule of interest and does not imply process limitations unless specified.

  Thienopyrrole compounds may be prepared as individual enantiomers or diastereomers, either in racemic form or by stereospecific synthesis or resolution. For example, compounds can be enantiomers or diastereomers of their components by standard methods, such as salt formation with an optically active base, followed by fractional crystallization and formation of stereoisomeric pairs by regeneration of the free acid. Can be divided into The compounds may be resolved by formation of stereoisomeric esters or amides, followed by chromatographic separation and removal of the chiral auxiliary. Alternatively, the compounds may be resolved using a chiral HPLC column. Enantiomers may be obtained from the kinetic resolution of racemic forms of the corresponding ester using a lipase enzyme.

Except where discussed below for tautomeric forms, structural (or constitutional) isomers (ie, isomerisms in which the bonds between the atoms are different, rather than merely differing by the location of the atoms in space) Body is specifically excluded from the term "isomer" as used herein. For example, a reference to a methoxy group, -OCH ₃ is a hydroxymethyl group which is a structural isomer, should not be construed as a reference to -CH ₂ OH. Similarly, a reference to ortho-chlorophenyl should not be construed as a reference to its structural isomer, meta-chlorophenyl. However, reference to a group of structures may also include structural isomeric forms belonging to that group (eg, C ₃ -alkyl or propyl includes n-propyl and isopropyl; C ₄ -alkyl or butyl is n-butyl, isobutyl, sec- and tert-butyl; methoxyphenyl includes ortho-methoxyphenyl, meta-methoxyphenyl and para-methoxyphenyl).

  The above exclusions are tautomers, eg keto, enol and enolate forms (eg, the following tautomers: keto / enol, imine / enamine, amide / iminoalcohol, amidine / amidine, nitroso Not applicable to (oximes, thioketones / enethiols, N-nitroso / hydroxyazos, and nitros / acyl-nitros).

It is particularly noted that compounds having one or more isotopic substituents are included in the term "isomer". For example, H may be present in any isotopic form such as ¹ H, ² H (D), and ³ H (T); C may be any such as ¹² C, ¹³ C, and ¹⁴ C O may exist in isotopic form; O may exist in any isotopic form such as ¹⁶ O and ¹⁸ O; and so forth.

3. Oplophorus Luciferase The thienopyrrole compounds of the invention may be used to inhibit Oplophorus-derived luciferase. Thienopyrrole compounds can inhibit the luciferase activity of Oplophorus-derived luciferase. The Oplophorus-derived luciferase may be a wild type Oplophorus luciferase, or a variant of Oplophorus luciferase, such as the luciferase of SEQ ID NO: 2. Oplophorus luciferase mutants are described in US Pat. Nos. 8,557,970 and 8,669,103, the entire contents of each of which are incorporated herein by reference.

  The polypeptide sequence of the mature 19 kDa subunit of the naturally occurring form of Oplophorus gracilirostris luciferase is provided in SEQ ID NO: 1. An exemplary polypeptide sequence of a synthetic Oplophorus-derived luciferase that can be used in the methods described herein is provided in SEQ ID NO: 2 (herein interchangeably “NanoLuc”, “Nluc” , "Nluc luciferase", and also "Nluc enzyme").

4. Coelenterazine Substrates The thienopyrrole compounds of the present invention may be used to inhibit luciferase activity by binding to luciferase and competing with or interfering with coelenterazine or coelenterazine derivative substrates. Coelenterazine substrates are a group of reporter molecules that emit cold light when acted upon by luciferase and other bioluminescent proteins. Examples of coelenterazine substrates include coelenterazine; coelenterazine derivatives and / or analogues, such as, for example, 2 in addition to those disclosed in WO 2003/040100, US Patent Publication No. 2008/0248511, and US Patent Publication No. US 2012/0117667. -Furanylmethyl-deoxy-coelenterazine (frimazine), coelenterazine-n, coelenterazine-f, coelenterazine-h, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, bis-deoxycoelenterazine (" Coelenterazine-hh "), coelenterazine-i, coelenterazine-icp, coelenterazine-v, and 2-methyl-coelenterazine; procoelenterazine (ie compounds Into a substrate for luciferase is not a substrate compound for non-luciferase), but Kinonmasuku been coelenterazine and the like, but are not limited to. Further examples of coelenterazine substrates are described, for example, in U.S. application 2012/0107849, U.S. application 2013/0130289, U.S. patent application 14 / 608,910, and U.S. patent application 14 / 609,372. Each of which is incorporated herein by reference.

5. Methods of Inhibiting Oplophorus Luciferase Activity Thienopyrrole compounds of the present disclosure can be used in methods of inhibiting Oplophorus luciferase activity. The method involves contacting a thienopyrrole compound disclosed herein (eg, a compound of Formula (I), (Ia), (Ib), or (Ib ′)) with cells expressing or containing an Oplophorus-derived luciferase The compounds of the present disclosure may selectively inhibit Oplophorus-derived luciferase. Thienopyrrole compounds of the present disclosure can be used in assays used to detect the presence or activity of an enzyme using Oplophorus luciferase to selectively inhibit the signal from Oplophorus luciferase. Samples, eg, proteins of interest (eg, enzymes, binding partners, ligands etc.), cofactors for enzymatic reactions, enzyme substrates, enzyme inhibitors, enzyme activators, or OH radicals, or one or more conditions, eg They may be used in bioluminescent methods employing Oplophorus luciferase and coelenterazine or coelenterazine derivative substrates to detect one or more molecules under redox conditions. While coelenterazine substrates act as substrates for Oplophorus luciferase, the claimed thienopyrrole compounds may be desirable in embodiments where such inhibition may be desired, for example, in applications involving time multiplexing of multiple bioluminescent systems, or In some plate-based luminescence assays, luciferase can be inhibited to act to selectively suppress the luminescent signal. For example, thienopyrrole compounds can be used to inhibit intracellular and / or extracellular Oplophorus luciferase activity.

(1) Thienopyrrole Compound As described above, the method comprises converting the thienopyrrole compound disclosed herein (eg, a compound of the formula (I), (Ia), (Ib) or (Ib ')) to an Oplophorus-derived luciferase The compound of the present disclosure can selectively inhibit Oplophorus-derived luciferase. In addition to the compounds of the formulas (I), (Ia), (Ib) and (Ib ′), the compounds which can be used in the methods described herein also include the compounds of the formula (II) or salts thereof :
(II)
During the ceremony:
The dotted lines indicate the presence or absence of a bond;
n is 0, 1, 2, 3, 4 or 5;
X is CH, N, O, or S;
When the dotted line represents the presence of a bond, X is CH or N, and when the dotted line represents the absence of a bond, X is O or S;
A is an optionally substituted phenyl ring or an optionally substituted 5 or 6 membered heteroaryl ring;
R ¹ and R ² are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally Selected from the group consisting of alkoxyalkyl substituted with and optionally substituted alkoxyalkoxyalkyl;
Z is selected from the group consisting of -NR ³ R ⁴ and -OR ⁵ ; and R ³ , R ⁴ and R ⁵ are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, Optionally substituted C ₃ -C ₈ -cycloalkyl, optionally substituted C ₃ -C ₈ -cycloalkylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted hetero Selected from the group consisting of aryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, and optionally substituted heterocyclylalkyl; or R ³ and R ⁴ with the nitrogen atom to which they are attached Together, form an optionally substituted ring together.

  In some embodiments, n is one.

  In some embodiments, the dotted line represents the presence of a bond and X is CH.

  In some embodiments, A is a phenyl ring. In some embodiments, A is a 5-membered heteroaryl ring. In some embodiments, A is a thienyl ring. In some embodiments, A is a furanyl ring.

In some embodiments, R ¹ is selected from the group consisting of hydrogen, C ₁ -C ₈ alkyl, halo-C ₁ -C ₈ -alkyl, alkoxyalkoxyalkyl, and arylalkyl. For example, in some embodiments, R ¹ is selected from the group consisting of hydrogen, ethyl, n-hexyl, 2- (2-methoxyethoxy) ethyl and benzyl. In some embodiments, R ¹ is ethyl.

In some embodiments, R ² is optionally substituted aryl. For example, in some embodiments, R ² is substituted phenyl. R ² is phenyl substituted with one substituent selected from the group consisting of C ₁ -C ₄ alkyl, cyano, amido, C ₁ -C ₄ alkoxy, and hydroxyalkyl. In some embodiments, R ² is phenyl substituted with one methyl group (eg, m-tolyl).

In some embodiments, R ¹ is C ₁ -C ₄ alkyl and R ² is optionally substituted phenyl. In some embodiments, R ¹ is ethyl and R ² is m-tolyl.

In some embodiments, Z is -NR ³ R ^4.

In some embodiments, R ³ and R ⁴ together with the nitrogen atom to which they are attached form an optionally substituted ring together. In some embodiments, R ³ and R ⁴ together with the nitrogen atom to which they are attached form an optionally substituted monocyclic heterocycle. In some embodiments, the optionally substituted monocyclic heterocycle is selected from the group consisting of optionally substituted pyrrolidine, piperidine and piperazine. In some embodiments, the optionally substituted monocyclic heterocycle is unsubstituted pyrrolidine, unsubstituted piperidine, piperidine substituted with one substituent (eg, alkoxycarbonyl such as ethoxycarbonyl), or 1 One of a substituent piperazines (eg, C ₁ -C ₄ alkyl such as methyl) is selected from the group consisting of.

In some embodiments, R ³ is hydrogen.

In some embodiments, R ⁴ is unsubstituted C ₁ -C ₈ alkyl (eg, methyl), carboxy-C ₁ -C ₈ -alkyl (eg,-(CH ₂ ) ₇ -COOH), C _1- C ₄ -Alkoxycarbonyl-C ₁ -C ₈ -alkyl (eg-(CH ₂ ) ₇ -COOCH ₃ ), optionally substituted phenyl (below), optionally substituted C ₅ -C ₆ cycloalkyl ( For example, alkoxycarbonylalkylcarbonyl such as unsubstituted cyclopentyl, unsubstituted cyclohexyl or substituted cyclohexyl), optionally substituted heterocyclyl (eg, unsubstituted piperidinyl, tert-butoxycarbonyl, -CO- (CH ₂ ) ₄ -COOCH _{3 and the} like or -CO- (CH _{2) 4} -COOH piperidinyl substituted with carboxyalkyl carbonyl, etc.), Ren In substituted heteroarylalkyl (e.g., -CH ₂ - pyridyl, such pyridyl -C ₁ -C ₄ - alkyl), and optionally substituted heterocyclylalkyl (for example, - (CH _{2) 3} - morpholino such morpholino -C ₁ -C ₄ - is selected from the group consisting of alkyl). For example, in some embodiments, R ⁴ is unsubstituted or C ₁ -C ₄ -alkoxycarbonyl (eg, -C (O) OCH ₃ ) and C ₁ -C ₄ -alkoxycarbonyl-C ₁ -C ₄ - alkyl _{(e.g., -CH 2 -C (O) CH} 2 CH 3) phenyl substituted with one substituent such as substituents selected from the group consisting of.

In some embodiments, R ⁴ is carboxy, C ₁ -C ₄ -alkoxycarbonyl (eg, -C (O) OCH ₃ ), C ₁ -C ₈ -alkylamide (eg, -C (O) NH 2 -(CH ₂ ) ₃ CH ₃ or -C (O) NH- (CH ₂ ) ₅ CH ₃ ), hydroxy-C ₁ -C ₈ -alkylamide (eg, -C (O) NH- (CH ₂ ) ₂ OH or _{-C (O) NH- (CH 2} ) 6 OH), amide (ie, -CONH _2), amino -C substituted with optionally ₁ -C ₈ - alkyl amide (e.g., -C (O) NH -(CH ₂ ) ₆ NH ₂ ), C ₁ -C ₄ -dialkylamino-C ₁ -C ₈ -alkylamide (eg -C (O) NH- (CH ₂ ) ₂ N (CH ₃ ) ₂ ), Carboxy-C ₁ -C ₈ -alkyl amides (eg, -C (O) NH- (CH ₂ ) ₃ CO OH, -C (O) NH- ( CH 2) 5 COOH or _{-C (O) NH- (CH 2} ) 7 COOH), sulfonic acid -C ₁ -C ₈ - alkyl amide (e.g., -C (O) _{NH- (CH 2) 6 SO 3} H), sulfonic acid -C ₁ -C ₈ - alkyl amide (e.g., -C (O) NH- (CH 2) 6 SO 3 -), C 1 -C 4 - alkyl Carbonyl-C ₁ -C ₈ -alkyl amides (eg, —C (O) NH— (CH ₂ ) ₃ COOCH ₃ , —C (O) NH— (CH ₂ ) ₅ COOCH ₃ or —C (O) NH— (CH ₂ ) ₇ COOCH ₃ ), optionally substituted C ₃ -C ₆ -cycloalkyl amides such as, for example, -C (O) NH-cyclohexyl, -C (O) NH-cyclohexyl-COOH or -C (O ) NH- cyclohexyl -COOCH _3), and Heteroshikurirua De (e.g., -C (O) NH- piperidinyl, unsubstituted or methyl substituted with tert- butoxycarbonyl or acetyl) cyclohexyl substituted with one substituent selected from the group consisting of. For example, in some embodiments, R ⁴ is optionally substituted amino-C ₁ -C ₈ -alkyl amide (eg, —C (O) NH— (CH ₂ ) ₆ NH ₂ ), amino The group is protected with an amino protecting group (e.g. tert-butoxycarbonyl) or the amino group is protonated to form a salt (e.g. a hydrochloride salt) or the amino group is a fluorophore (e.g. fluorescein) Or polypeptide (eg -Asp-Asp-Asp peptide, optionally acetylated at the end).

In some embodiments, Z is -OR ⁵ . In some embodiments, R ⁵ is H. In some embodiments, R ⁵ is optionally substituted C ₁ -C ₄ alkyl (eg, methyl).

(2) Use of Cell Impermeable Thienopyrrole Compounds In certain embodiments, the methods disclosed herein modify a sample (e.g., a cell) with a cell permeable coelenterazine substrate to be cell impermeable Contacting with a mixture with a compound described herein. In such embodiments, the thienopyrrole compounds and methods of the present disclosure may be used to selectively inhibit any luciferase that may be excreted from cells after constructing the initial brightness of a high throughput screening procedure assay format, The emission that may occur outside may be selectively inhibited. Such methods can provide more selective signals in cells. Examples of cell impermeable thienopyrrole compounds include JRW-0051, JRW-0147, and JRW-0187.

(3) Use of Cell Permeable Thienopyrrole Compounds In certain embodiments, the methods disclosed herein are cell permeable to a sample (eg, a cell) and a cell permeable coelenterazine substrate as described herein. Contacting with a mixture with a compound of In such embodiments, thienopyrrole compounds of the present disclosure can enter cells and selectively inhibit Oplophorus luciferase therein. Such methods are advantageous in multiplex assays involving the use of two or more luciferases and can inhibit the luminescence from Oplophorus luciferase so that the luminescence from another luciferase in the cell is selectively viewed. obtain. Examples of cell permeable thienopyrrole compounds include JRW-0013 and JRW-0138.

(4) Use with Transcription Reporter The thienopyrrole compounds of the present disclosure may be used with gene transcription reporter systems. In certain embodiments, provided is a method of measuring the activity of a promoter in a sample, wherein the promoter is operably linked to a gene encoding an Oplophorus-derived luciferase or a variant thereof. The method comprises (a) contacting the sample with a coelenterazine substrate and (b) determining the activity of the promoter by measuring the luminescence of the sample, wherein the sample comprises the promoter. The method may further comprise the step of contacting the sample with a thienopyrrole compound as described herein to selectively inhibit luminescence. The promoter may be operably linked to the gene via translational fusion or transcriptional fusion. The biological pathway of interest may be examined, for example, by treating cells containing a promoter operably linked to the gene encoding luciferase with an inducer of the pathway. This promoter activity is then measured and monitored to examine any correlation between the activity of the promoter and the pathway, as well as to obtain kinetic measurements related to gene expression (e.g. It is also good. Luminescence can be selectively inhibited using the thienopyrrole compounds described herein.

(5) Multiplexing Thienopyrrole compounds of the present disclosure may be used to inhibit Oplophorus luciferase when applied to time multiplexing with other luciferases and assays. In some embodiments, the Oplophorus-derived luciferase or variant thereof is another enzyme that emits light at a different wavelength (eg, luciferase), eg, green firefly luciferase, eg, Photinus pyralis (eg, Luc2; Promega Corp) or red It may be multiplexed with click beetle luciferase (CHROMA-LUCTM luciferase; Promega Corp.). For example, if Oplophorus luciferase is used as a functional reporter, then green firefly luciferase or red CHROMA-LUCTM luciferase may be used to control non-specific effects on gene regulation, Alternatively, the transfection efficiency may be normalized. In some embodiments, the emission generated from Oplophorus luciferase (approximately 460 nm) and the emission generated from red CHROMA-LUCTM (approximately 610 nm) can be easily resolved using a luminometer with a wavelength discrimination filter And allows measurement of both signals from the same sample. In such embodiments, the Thienopyrrole Compounds described herein can be used to selectively inhibit Oplophorus luciferase so that signals from other luciferases can be selectively viewed.

  In another example, Oplophorus luciferase may be used as a transcription reporter, paired with luciferases that emit light at different wavelengths contained in the assay reagent. In another example, Oplophorus luciferase may be used with one or more additional luciferases, wherein the luminescence of each luciferase may be measured separately through the use of selective enzyme inhibitors. For example, the luminescence of Oplophorus luciferase may be measured upon addition of a suitable substrate and buffer, as described herein that is selective to the suitable substrate and buffer and Oplophorus luciferase. Measurement of the second luciferase upon subsequent addition of the thienopyrrole compound may follow. In another example, the Oplophorus luciferase contained in the assay reagent may be used in certain aspects of cell physiology, eg, to measure ATP to estimate cell viability, or to measure caspase activity. May be used to estimate cell apoptosis.

(6) Bioluminescence resonance energy transfer (BRET)
The thienopyrrole compounds of the present disclosure may be used in any method where Oplophorus luciferase is used to detect ligand-protein and / or protein-protein interactions. In various embodiments, Oplophorus luciferase may be used to transfer energy to an energy acceptor. One such method is bioluminescence resonance energy transfer (BRET). For BRET, energy transfer from the bioluminescent donor to the fluorescent acceptor produces a shift in the spectral distribution of the emission. This energy transfer allows real-time monitoring of protein-protein or ligand-protein interactions in vitro or in vivo. In some embodiments, the BRET method may be a Nluc-mediated bioluminescence resonance energy transfer (such as NanoBRET) assay for ligand-protein and / or protein-protein interactions. NANOBRET uses two different methods: 1) HALOTAG and Nluc based techniques, using bioluminescence resonance energy transfer (BRET) to detect protein-protein and / or ligand-protein interactions, with increased signal 2) may be achieved by reduced spectral overlap; and 2) detecting ligand-receptor interaction in living cells with Nluc luciferase fused to the protein of interest and a fluorescent tracer.

  In some embodiments, used in BRET analysis (ie, Oplophorus luciferase), can two molecules be able to bind to one another or co-localize in cells You can decide if there is. For example, a first fusion protein can be created using a luminescent enzyme as a bioluminescent donor molecule in combination with a molecule of interest or a protein of interest. In various embodiments, the first fusion protein comprises a luminescent enzyme and a protein of interest. In various embodiments, a first fusion protein containing a luminescent enzyme is used for BRET analysis to determine whether the protein / protein is in a system including, but not limited to, cell lysates, intact cells and live animals. Interactions can be detected. In various embodiments, HALOTAG can be used as a fluorescent acceptor molecule. In some embodiments, HALOTAG can be fused to a second protein of interest or a luminescent enzyme. For example, the luminescent enzyme can be fused to HALOTAG, expressed in cells or animals, and labeled with a fluorescent HALOTAG ligand such as HALOTAG TMR ligand. The fusion can then be excited to fluoresce in the presence of a cell permeable luminescent enzyme substrate. In some embodiments, fluorescent proteins, including but not limited to green fluorescent protein (GFP) or red fluorescent protein (RFP), to name a few non-limiting examples, fluorescein, rhodamine green, oregon green, Alternatively, BRET may be performed using a luminescent enzyme in combination with a fluorescent label comprising Alexa 488.

  In some embodiments, quenching the signal from excreted Nluc can improve the signal to noise ratio when using the NanoBRET plate assay.

  In certain embodiments, cell permeable thienopyrrole compounds may be used to inhibit intracellular BRET. In certain embodiments, cell impermeable thienopyrrole compounds may be used to inhibit extracellular BRET. In certain embodiments, cell impermeable thienopyrrole compounds may be used in a target association model.

(7) Protein Proximity Assay for Living Cells or Lytic Forms In some embodiments, Oplophorus luciferase is used in conjunction with a circulating replacement (CP) or linear cleavage (SS) luminescent enzyme fusion protein to provide proximity of a protein. May be measured. Oplophorus luciferase is substituted or cleaved through insertion of the amino acid sequence (eg, TEV) of the protease substrate to generate low bioluminescence. Inactive luciferase is linked to the monitor protein (eg, via gene fusion). Potentially interacting proteins are tethered (eg, via gene fusion) to a protease (eg, TEV). When the two monitor proteins interact or are in close proximity (eg, via constitutive interactions, drug stimulation or pathway responses), the luminescent enzyme is cleaved to produce high bioluminescent activity. The example may be applied to the measurement of protein proximity in cell or biochemical assays.

(8) Protein Complementation Assay In some embodiments, the thienopyrrole compounds of the present disclosure detect ligand-protein and protein-protein interactions or proximity such as, for example, protein complementation assay (PCA) or enzyme fragmentation assay If such luciferases are used in other ways, they may be used to inhibit Oplophorus luciferase. Protein complementation assay (PCA) provides a means to detect the interaction of two biomolecules, eg, polypeptides. PCA utilizes two fragments of the same protein, eg, an enzyme, which can be reconstituted into a functionally active protein when in close proximity to each other. In some embodiments, NANOBIT® technology (Promega Corporation) may be used to detect the proximity of molecules by the reconstitution of the luminescent enzyme through the binding interaction of the components or subunits of the enzyme . NANOBIT utilizes the Oplophorus luciferase mutant, a non-luminescent peptide (NLPep) derived from Nluc luciferase and a non-luminescent polypeptide (NL Poly). NLPep and NLPoly are fused to a protein of interest. If the protein of interest interacts, NLPep and NLPoly interact to reconstitute the full-length Oplophorus luciferase enzyme.

  For example, the luminescent enzyme can be separated into two fragments at a site that is tolerant of separation, and the pair of target polypeptides considered to interact with each fragment of the separated luminescent enzyme, eg, one of FKBP and FRB It can be fused. If the two polypeptides of interest actually interact, for example, the fragments of the luminescent enzyme will then be in close proximity to one another to reconstitute a functional, active luminescent enzyme. In some embodiments, the activity of the reconstituted luminescent enzyme can then be detected and measured. In some embodiments, lac-Z (Langley et al., PNAS 72: 1254-1257 (1975)) or ribonuclease S (Levit and Berger, J. Biol. Chem. 251: 1333-1339 (1976)). The split luminescent enzyme can be used in a similar more general complementary system. In some embodiments, a luminescent enzyme fragment ("A") known to be complementary to another luminescent enzyme fragment (designated "B") can be fused to the target protein and contain fragment B. The resulting fusion can be monitored via luminescence in the cells or cell lysates. In some embodiments, the source of fragment B may be the same cell (eg, if the gene for fragment B is integrated into the cell's genome or contained in another plasmid in the cell) Or it may be a lysate or purified protein from another cell. In some embodiments, this same fusion protein (fragment A) is captured or immobilized using a fusion between a polypeptide such as HALO TAG capable of attachment to a solid support and fragment B It is also good. In some embodiments, luminescence can be used to reveal a successful capture or to quantify the amount of material captured.

(9) Dimerization Assay In some embodiments, the thienopyrrole compounds of the present disclosure are used in conjunction with full-length, cyclically substituted luminescent enzymes fused to their respective binding partners, eg, FRB and FKBP, and are protein-complementary It may be used in an assay. An important difference between the methods disclosed herein and conventional protein complementarity was that there was no complementarity, but rather, a dimer of two full-length enzymes, eg, a cyclic substitution luminescent enzyme That there was a

  Briefly, a circulating replacement reporter protein similarly constructed for low activity is fused to both of the fusion protein partners. For example, each fusion partner may be linked to the identically constructed replacement reporter. The interaction of the fusion partners brings the replacement reporters closer together, thereby allowing the reconstitution of hybrid reporters with higher activity.

6. Samples The thienopyrrole compounds of the present disclosure may be used with samples containing biological components. The sample may comprise cells. The sample may be a heterogeneous mixture of components (including intact cells, cell extracts, cell lysates, bacteria, viruses, organelles, exosomes, and mixtures thereof) or single components or components (eg, natural or It may comprise a homogeneous group of synthetic amino acids, nucleic acids or carbohydrate polymers, or lipid membrane complexes). Thienopyrrole compounds can generally be nontoxic to living cells and other biological components within the concentration of use.

  The sample may be animal (eg, vertebrate), plant, fungus, physiological fluid (eg, blood, plasma, urine, mucus, secretion etc.), cell, cell lysate, cell supernatant, or purified fraction of cell (Eg, subcellular fractions) may be included. In certain embodiments, the sample may be a cell. In some embodiments, the sample may be a living cell. Cells include, but are not limited to, eukaryotic cells such as, for example, yeast, avian, plant, insect cells or human, monkey, mouse, dog, bovine, equine, feline, ovine, goat, or porcine cells. It may be a mammalian cell, or a prokaryotic cell, or a cell derived from two or more different organisms, or a cell lysate or its supernatant. The cells may not be genetically engineered via recombinant technology (non-recombinant cells), or may be transiently transfected with recombinant DNA, and / or their genomes stabilized by recombinant DNA. Recombination in which the genome is modified to enhance the gene or to disrupt the gene, eg to destroy the promoter, intron or open reading frame, or to replace one of the DNA fragments with another It may be a cell. The recombinant DNA or fragment of the replacement DNA is independent of the molecule to be detected by the method of the invention, the part that alters the level or activity of the molecule to be detected, and / or the molecule or part that alters the level or activity of the molecule It may encode a gene product. The cells may or may not express luciferase. The cells may be genetically engineered via recombinant technology.

7. Kits Disclosed are kits for measuring the presence or activity of one or more enzymes (eg, Oplophorus or Oplophorus mutant luciferases). The kit comprises the following: Oplophorus or Oplophorus mutant luciferase, coelenterazine or coelenterazine derivative substrate, a compound or composition of the invention capable of inhibiting Oplophorus or Oplophorus mutant luciferase, instructions for performing a luminescence assay, and a reaction It may also contain one or more buffers. The reaction buffer may be present in separate formulations for non-luciferase and luminescent enzyme reactions or may be present in a single formulation for single step assays. The kit may also contain other inhibitors, activators, and / or enhancers for non-luciferase enzymes. The kit may also contain positive and / or negative controls for the assay.

8. EXAMPLES General Synthesis Procedure A: To a solution of the ester intermediate (1 equivalent) in dioxane / water (4: 1) was added lithium hydroxide (5 equivalents). The suspension was heated to 60 ° C. (monitored by LCMS or TLC analysis) until the starting material was consumed. The reaction mixture was cooled and acidified to pH 3 with HCl (2 M). The suspension was partitioned between ethyl acetate and water. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue is purified by column chromatography (silica, dichloromethane / methanol) to give the desired product.

  General Synthetic Procedure B: A solution of carboxylic acid intermediate (1 equivalent) in dimethylformamide with the required amine (or amine hydrochloride), hydroxybenzotriazole (2 equivalents), 1-ethyl-3- (3-dimethyl) Aminopropyl) carbodiimide (2 equivalents) and diisopropylethylamine (3 equivalents) were added. The mixture was heated to 60 ° C. (monitored by LCMS or TLC analysis) until the starting material was consumed. The reaction mixture was cooled, diluted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue is purified by column chromatography (silica, dichloromethane / methanol or heptane / ethyl acetate) to give the desired product.

Example 1
General Synthesis of Compounds of Formula (Ia) Compounds of formula (Ia) can be generally synthesized according to Scheme 1.
Scheme 1.
2-Chloro-N-ethyl-N- (m-tolyl) acetamide (JRW-0003)

Water (12 mL) was added to a solution of N-ethyl-3-methylaniline (2.0 g, 14.8 mmol) in ethyl acetate (25 mL). The biphasic solution was cooled to 0 ° C. and potassium hydroxide (2.49 g, 44.4 mmol) was added in one portion. 2-Chloroacetyl chloride (2.5 g, 1.8 mL, 22.2 mmol) was added dropwise over 10 minutes. The mixture was stirred for 1 hour, diluted with water and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated to give the crude product (3.2 g) as mobile oil. ESI MS m / z 212 [M + H] ^< +>.
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate methyl (JRW-0004)

To a solution of 2-chloro-N-ethyl-N- (m-tolyl) acetamide (14.8 mmol) in acetonitrile (100 mL), methyl 4H-thieno [3,2-b] pyrrole-5-carboxylate (2 .28 g, 12.6 mmol), potassium carbonate (2.09 g, 15.1 mmol) and 18-crown-6 (166 mg, 0.63 mmol) were added. The mixture was heated to reflux for 5 hours and the reaction was concentrated to about 20 mL volume under vacuum. The suspension was diluted with water, filtered and washed with water. The solid was dried under vacuum to give the crude product (4.6 g) as a pale brown solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.53 (d, J = 5.4, ¹ H), 7.46-7.36 (m, 1 H), 7.35-7.11 (m, 5H), 4.92 (s, 2H), 3.74 (s, 3H), 3.67-3.53 (m, 2H), 2.37 (s, 3H), 1.00 (t, J = 6.6, 3 H); ESI MS m / z 357 [M + H] ^< +>.
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (WZ-141-74)

Following general procedure A, methyl 4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate (2.0 g, 5 .6 mmol) was reacted with lithium hydroxide (671 mg, 28.0 mmol) to give the desired product (1.8 g, 93%) as a pale yellow solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 12.45 (s, 1 H), 7.49 (d, J = 5.4, 1 H), 7.44-7.37 (m, 1 H), 7 .33-7.12 (m, 4H), 7.09 (s, 1 H), 4.91 (s, 2 H), 3.71-3.54 (m, 2 H), 2.36 (s, 3 H) ), 1.08-0.94 (s, 3 H); ESI MS m / z 343 [M + H] ^< +>.
Example 2
N-Cyclohexyl-4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide (JRW-0006)

4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (75 mg, 0.22 mmol) according to General Procedure B Was reacted with cyclohexylamine (43 mg, 0.44 mmol) to give the desired product (50 mg, 54%) as a white solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.83 (d, J = 8. ^{1, 1} H), 7.44-7.16 (m, 5 H), 7.13-7.06 (m, 5-7) 2H), 4.96 (s, 2H), 3.75-3.52 (m, 2H), 2.36 (s, 3H), 1.84-1.51 (m, 5H), 1.37 -1.17 (m, 6H), 1.05-0.93 (m, 3H); ESI MS m / z 424 [M + H] +; HPLC> 99% (AUC), T R 7.02 min; UV (MeOH) λ _max 289 nm, ε 25, 200.
Example 3
N-ethyl-2- (5- (pyrrolidine-1-carbonyl) -4H-thieno [3,2-b] pyrrol-4-yl) -N- (m-tolyl) acetamide (JRW-0008)

4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (75 mg, 0.22 mmol) according to General Procedure B Was reacted with pyrrolidine (43 mg, 0.44 mmol) to give the desired product (70 mg, 81%) as an off white solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.45-7.16 (m, 5 H), 7.06 (d, J = 5.2, 1 H), 6.83 (s, 1 H), 4 .83 (s, 2 H), 3.77-3.32 (m, 6 H), 2.36 (s, 3 H), 1.91-1.75 (m, 4 H), 1.06-0.92 (m, 3H); ESI MS m / z 396 [M + H] +; HPLC97.3% (AUC), T R 6.24 _{min; UV (MeOH) λ max 288nm} , ε21,773.
Example 4
N-ethyl-2- (5- (piperidine-1-carbonyl) -4H-thieno [3,2-b] pyrrol-4-yl) -N- (m-tolyl) acetamide (JRW-0009)

4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (80 mg, 0.23 mmol) according to General Procedure B Was reacted with piperidine (40 mg, 0.47 mmol) to give the desired product (90 mg, 94%) as an orange gum. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.52-7.13 (m, 5 H), 7.05 (d, J = 5.2, 1 H), 6.58 (s, 1 H), 4 .75 (s, 2 H), 3.68-3.45 (m, 6 H), 2.37 (s, 3 H), 1.69-1.42 (m, 6 H), 1.00 (t, J = 6.9,3H); ESI MS m / z 410 [M + H] +; HPLC98.8% (AUC), T R 5.91 _{min; UV (MeOH) λ max 284nm} , ε24,598.
Example 5
1- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carbonyl) piperidine-4-carboxylate (JRW-0012 )

4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (120 mg, 0.35 mmol) according to General Procedure B Was reacted with ethyl piperidine-4-carboxylate (110 mg, 0.70 mmol) to give the desired product (160 mg, 94%) as a white foam. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.47-7.36 (m, 1 H), 7.32 (d, J = 5.3, ¹ H), 7.29-7.14 (m, 3H), 7.07 (d, J = 5.3, 1 H), 6.61 (s, 1 H), 4. 75 (s, 2 H), 4.22 (d, J = 13.2, 2 H) , 4.06 (q, J = 7.1, 2H), 3.68-3.52 (m, 2H), 3.18-2.94 (m, 2H), 2. 70-2.55 ( m, 1H), 2.37 (s, 3H), 1.90-1.78 (m, 2H), 1.62-1.48 (m, 2H), 1.17 (t, J = 7). 1,3H), 0.98 (t, J = 6.8,3H); ESI MS m / z 482 [M + H] +; HPLC> 99% (AUC), T R 7.25 min; UV (MeOH) λ _max 286nm, ε20,00 .
Example 6
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N-phenyl-4H-thieno [3,2-b] pyrrole-5-carboxamide (JRW-0143)

4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (50 mg, 0.15 mmol) according to General Procedure B Was reacted with aniline (16 mg, 0.18 mmol) to give the desired product (30 mg, 49%) as a foam. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 9.93 (s, 1 H), 7.74-7.68 (m, 2 H), 7.49-7. 19 (m, 9 H), 7.16 (D, J = 5.3, 1 H), 7.05 (t, J = 7.4, 1 H), 4.99 (s, 2 H), 3.67-3.52 (m, 2 H), 2 .37 (s, 3H), 1.06-0.92 (m, 3H); ESI MS m / z 418 [M + H] +; HPLC85.4% (AUC), T R 6.04 min; UV (EtOH ) Λ _max 306 nm, ε 27, 330.
Example 7
Ethyl 2- (4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) phenyl) acetate (JRW-0152 )

4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (50 mg, 0.15 mmol) in DMF (3 mL) To the solution was added ethyl 2- (4-aminophenyl) acetate (31 mg, 0.18 mmol), HATU (111 mg, 0.29 mmol) and diisopropylethylamine (56 mg, 0.44 mmol). The reaction was heated to 60 ° C. for 18 hours. The reaction mixture was cooled, diluted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue is purified by column chromatography (silica, heptane / ethyl acetate) to give the desired product (28 mg, 38%) as an orange solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 9.93 (s, 1 H), 7.64 (d, J = 7.6, 2 H), 7.50-7.11 (m, 9 H), 4 .99 (s, 2H), 4.06 (q, J = 6.4 Hz, 2H), 3.60 (s, 4H), 2.37 (s, 3H), 1.17 (t, J = 7.2,3H), 0.98 (t, J = 6.4,3H); ESI MS m / z 476 [M + H] +; HPLC97.2% (AUC), T R 7.61 min; UV ( EtOH) λ _max 308 nm, ε 34, 350.
Example 8
Methyl 3- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) benzoate (JRW-0151)

4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (50 mg, 0.15 mmol) in DMF (3 mL) To the solution was added methyl 3-aminobenzoate (33 mg, 0.22 mmol), HATU (111 mg, 0.29 mmol) and diisopropylethylamine (56 mg, 0.44 mmol). The reaction was heated to 60 ° C. for 18 hours. The reaction mixture was cooled, diluted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue was purified by column chromatography (silica, heptane / ethyl acetate) to give the desired product (30 mg, 43%) as a pale yellow solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 10.15 (s, 1 H), 8.42-8. 38 (m, 1 H), 8.00 (d, J = 8.2, 1 H), 7 .67-7.63 (m, 1 H), 7.56-7. 10 (m, 8 H), 5.00 (s, 2 H), 3.86 (s, 3 H), 3.67-3.55 (M, 2 H), 2. 38 (s, 3 H), 0.99 (t, J = 6.7, 3 H); ESI MS m / z 476 [M + H] ⁺ ; HPLC 98.3% (AUC), T _R 7.52 min; UV (EtOH) λ _max 309 nm, ε 37,302.
Example 9
Methyl-cis-4- (4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1-carboxylic acid JRW-0041)

4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (150 mg, 0.44 mmol) according to General Procedure B Was reacted with cis-4-aminocyclohexane-1-carboxylic acid methyl hydrochloride (127 mg, 0.66 mmol) to give the desired product (186 mg, 88%) as a white foam. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.82 (d, J = 7.8, ¹ H), 7.45 to 7.33 (m, 2 H), 7.32 to 7.16 (m, 7) 3H), 7.12 (s, 1 H), 7.08 (d, J = 5.3, 1 H), 4.95 (s, 2 H), 3.85-3. 70 (m, 1 H), 3 .68-3.53 (m, 5 H), 2.63-2.56 (m, 1 H), 2.36 (s, 3 H), 2.08-1.92 (m, 2 H), 1.69 -1.43 (m, 6H), 1.08-0.95 (m, 3H); ESI MS m / z 482 [M + H] +; HPLC> 99% (AUC), T R 7.16 min; UV (MeOH) λ _max 288 nm, ε 24 998.
Example 10
6- (cis-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxamide) Hexanoic acid (JRW-0264)
Step 1. Cis-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid (JRW- 0261)

According to the general procedure A, methyl-cis-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) Cyclohexane-1-carboxylic acid (160 mg, 0.33 mmol) was reacted with lithium hydroxide (40 mg, 1.66 mmol) to give the crude product as a pale brown solid. ESI MS m / z 468 [M + H] ^< +>.
Step 2. 2- (cis-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1 -Carboxamide) methyl hexanoate (JRW-0262)

Following the general procedure B, cis-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane The 1-carboxylic acid (68 mg, 0.15 mmol) was reacted with 6-aminohexanoic acid methyl hydrochloride (40 mg, 0.22 mmol) to give the desired product (63 mg, 72%) as a pale red foam. ESI MS m / z 595 [M + H] ^< +>.
Step 3.6- (cis-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1 -Carboxamide) hexanoic acid (JRW-0264)

6- (cis-4- (4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) according to General Procedure A )) Cyclohexane-1-carboxamido) Methyl hexanoate (60 mg, 0.10 mmol) was reacted with lithium hydroxide (12 mg, 0.50 mmol) to give the desired product (56 mg, 95%) as a pale red foam . ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 11.96 (s, 1 H), 7.77 (d, J = 7.2, ¹ H), 7.61 (t, J = 5.5, 1 H) , 7.45-7.33 (m, 2H), 7.33-7.14 (m, 4H), 7.08 (d, J = 5.5, 1 H), 4.95 (s, 2 H) , 3.92-3.74 (m, 1 H), 3.67-3.56 (m, 2 H), 3.01 (dd, J = 6.8, 12.6, 2 H), 2.36 s, 3H), 2.27-2.10 (m, 3H), 1.94 to 1.79 (m, 2H), 1.78 to 1.64 (m, 2H), 1.60-1. 31 (m, 8 H), 1.30-1. 18 (m, 2 H), 1.06-0.94 (m, 3 H). ; ESI MS m / z 581 [ M + H] +; HPLC97.8% (AUC), T R 5.50 _{min; UV (EtOH) λ max 289nm} , ε18,873.
Example 11
8- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) octanoic acid (JRW-0198)
Step 1.8- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) methyl octanoate (JRW-0196)

4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (50 mg, 0.15 mmol) according to General Procedure B Was reacted with methyl 8-aminooctanoate hydrochloride (46 mg, 0.22 mmol) to give the desired product (72 mg, 99%) as an oil. ESI MS m / z 498 [M + H] ^< +>.
Step 2.8-(4- (2- (ethyl (m- tolyl) amino) -2- oxoethyl)-4 H-thieno [3, 2- b] pyrrole -5- carboxamido) octanoic acid (JRW- 0198)

Following the general procedure A, methyl 8- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) octanoate ( 72 mg, 0.14 mmol) was reacted with lithium hydroxide (17 mg, 0.72 mmol) to give the desired product (47 mg, 67%) as a white foam. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 11.91 (s, 1 H), 8.09 (t, J = 5.3, ¹ H), 7.47-7.16 (m, 5 H), 7 .09 (d, J = 5.3, 1 H), 7.03 (s, 1 H), 4.96 (s, 2 H), 3.72-3.52 (m, 2 H), 3.16 (dd , J = 6.4, 12.8, 2H), 2.36 (s, 3H), 2.17 (t, J = 7.3, 2H), 1.60-1.40 (m, 4H) , 1.36-1.18 (m, 6H), 1.08-0.93 (m, 3H); ESI MS m / z 484 [M + H] +; HPLC99.4% (AUC), T R 5. 43 min; UV (EtOH) λ _max 288 nm, ε 24, 627.
Example 12
6- (4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) piperidin-1-yl) -6 -Oxohexanoic acid (JRW-0208)
Step 1. tert-Butyl 4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) piperidine-1-carboxylic acid (JRW -0203)

4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (75 mg, 0.22 mmol) according to General Procedure B Was reacted with tert-butyl 4-aminopiperidine-1-carboxylate (66 mg, 0.33 mmol) to give the desired product (120 mg, quant) as a white foam. ESI MS m / z 525 [M + H] ^< +>.
Step 2. 4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (piperidin-4-yl) -4H-thieno [3,2-b] pyrrole-5-carboxamide (JRW -0204)

4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) piperidine-1-carvone in dichloromethane (5 mL) To a solution of acid tert-butyl (120 mg, 0.22 mmol) was added trifluoroacetic acid (1 mL). The reaction was stirred at room temperature for 1 hour. The mixture was diluted with toluene and concentrated in vacuo (3 ×) to give a pale brown oil crude product (170 mg). ESI MS m / z 425 [M + H] ^< +>.
Step 3.6 4- (4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) piperidin-1-yl ) Methyl -6-oxohexanoate (JRW-0206)

4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (piperidin-4-yl) -4H-thieno [3,2-b in dichloromethane (5 mL) cooled to 0 ° C. To a solution of pyrrole-5-carboxamide (0.22 mmol) was added methyl 6-chloro-6-oxohexanoate (49 mg, 0.27 mmol) and diisopropylethylamine (147 mg, 1.1 mmol). The reaction was stirred at 0 ° C. for 30 minutes. The mixture was diluted with dichloromethane and water and the layers separated. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated. The residue is purified by column chromatography (silica, dichloromethane / methanol) to give the desired product (100 mg, 77%) as a white foam. ESI MS m / z 567 [M + H] ^< +>.
Step 4. 4- (4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) piperidin-1-yl ) -6-Oxohexanoic acid (JRW-0208)

6- (4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) piperidine according to General Procedure A Methyl -1-yl) -6-oxohexanoate (100 mg, 0.18 mmol) is reacted with lithium hydroxide (21 mg, 0.88 mmol) to give the desired product (98 mg, quant.) As a white foam. The ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 11.96 (s, 1 H), 7.93 (d, J = 7.9, 1 H), 7.46-7.34 (m, 2 H), 7 .33-7.17 (m, 3 H), 7.12-7.06 (m, 2 H), 4.96 (s, 2 H), 4. 39-4.26 (m, 1 H), 4.05 -3.75 (m, 2 H), 3.68-3.54 (m, 2 H), 3.17-3.00 (m, 1 H), 2.73-2.57 (m, 1 H), 2 .42-2. 25 (m, 5 H), 2.24-2.12 (m, 2 H), 1.86-1.67 (m, 2 H), 1.55-1.11 (m, 6 H) , 1.07-0.93 (m, 3H); ESI MS m / z 553 [M + H] +; HPLC98.8% (AUC), T R 5.30 _{min; UV (MeOH) λ max 289nm} , ε23, 0 5.
Example 13
6- (trans-4-((4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) methyl) cyclohexane-1 -Carboxamide) methyl hexanoate (JRW-0267)

4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (133 mg, 0.39 mmol) according to General Procedure B Was reacted with methyl 6- (trans-4- (aminomethyl) cyclohexane-1-carboxamido) hexanoate (111 mg, 0.39 mmol) to give the desired product (180 mg, 76%) as a white foam. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 8.09 (t, J = 5.6, ¹ H), 7.61 (t, J = 5.5, ¹ H), 7.66-7.56 ( m, 2H), 7.32-7.16 (m, 3H), 7.14-6.99 (m, 2H), 4.96 (s, 2H), 3.67-3.46 (m, 2) 5H), 3.07-2.92 (m, 4H), 2.37 (s, 3H), 2.25 (t, J = 7.4, 2H), 2.08-1.93 (m, 5) 1 H), 1.83 to 1.60 (m, 4 H), 1.56 to 1.12 (m, 10 H), 1.08 to 0.80 (m, 5 H); ESI MS m / z 609 [M + H ^{] +; HPLC> 99% (} AUC), T R 5.95 _{min; UV (EtOH) λ max 288nm} , ε20,078.
Example 14
6- (trans-4-((4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) methyl) cyclohexane-1 -Carboxamide) Hexanoic acid (JRW-0268)

Following general procedure A, 6- (trans-4-((4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-) Carboxamido) methyl) cyclohexane-1-carboxamido) methyl hexanoate (165 mg, 0.27 mmol) is reacted with lithium hydroxide (32 mg, 3.4 mmol) to give the desired product (115 mg, 71%) as a white foam Obtained. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 11.93 (s, 1 H), 8.14-8.04 (m, 1 H), 7.61 (t, J = 5.3, 1 H), 7 .46-7.16 (m, 5 H), 7.13-6.98 (m, 2 H), 4.96 (s, 2 H), 3.68-3.53 (m, 2 H), 3.08 -2.92 (m, 4 H), 2.36 (s, 3 H), 2. 15 (t, J = 7.2, 2 H), 2.07-1.92 (m, 1 H), 1.80 -1.63 (m, 4H), 1.54-1.14 (m, 10H), 1.08-0.79 (m, 5H); ESI MS m / z 595 [M + H] ⁺ ; HPLC> 99 % (AUC), T _R 5.44 _{min; UV (EtOH) λ max 289nm} , ε25,604.
Example 15
General synthesis of compounds of formula (Ib)

Compounds of formula (Ib) can be synthesized generally according to scheme 2.
Scheme 2.
Example 16
Trans-Methyl-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1-carboxylic acid JRW-0013)

4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (100 mg, 0.29 mmol) according to General Procedure B Was reacted with methyl trans-4-aminocyclohexane-1-carboxylate (84 mg 0.44 mmol) to give the desired product (103 mg, 73%) as a white foam. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.89 (d, J = 7.9, ¹ H), 7.46-7.36 (m, 2 H), 7.34-7.15 (m, 7) 3H), 7.12-7.04 (m, 2H), 4.96 (s, 2H), 3.72-3.46 (s, 6H), 2.36 (s, 3H), 2. 32 -2.18 (m, 1 H), 2.03-1.77 (m, 4 H), 1.52-1.20 (m, 4 H), 1.06-0.95 (m, 3 H); ESI MS m / z 482 [M + H] ⁺ ; HPLC 99.0% (AUC), T _R 7.75 min; UV (MeOH) λ _max 289 nm, ε 26, 100.
Example 17
Trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid (JRW- 0034)

According to the general procedure A, trans-methyl-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) Cyclohexane-1-carboxylic acid (55 mg, 0.11 mmol) was reacted with lithium hydroxide (14 mg, 0.57 mmol) to give the desired product (50 mg, 93%) as a white solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 11.99 (s, 1 H), 7.92-7.84 (m, 1 H), 7.44-7.19 (m, 5 H), 7.12 −7.04 (m, 2 H), 4.97 (s, 2 H), 3.73-3.52 (m, 3 H), 2.36 (s, 3 H), 2.22-2.06 (m , 1 H), 1.98-1.76 (m, 4 H), 1.47-1.21 (m, 4 H), 1.08-0.93 (m, 3 H); ESI MS m / z 468 [ ^{M + H] +; HPLC99.4%} (AUC), T R 5.81 _{min; UV (MeOH) λ max 290nm} , ε26,502.
Example 18
N- (trans-4- (butylcarbamoyl) cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide ( JRW-0042)

According to the general procedure B, trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane The 1-carboxylic acid (50 mg, 0.11 mmol) was reacted with butylamine (9 mg, 0.13 mmol) to give the desired product (50 mg, 89%) as a white solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.82 (d, J = 8. ^{1, 1} H), 7.65-7.57 (m, 1 H), 7.46-7.11 (m, 1 H) 5H), 7.06 to 6.98 (m, 2H), 4.91 (s, 2H), 3.66 to 3.45 (m, 3H), 2.96 (q, J = 6.0 Hz) , 2H), 2.32 (s, 3H), 2.05 to 1.92 (m, 1H), 1.85 to 1.60 (m, 4H), 1.50 to 1.10 (m, 9H) ), 1.02-0.90 (m, 3 H), 0.80 (t, J = 7.2, 3 H); ESI MS m / z 523 [M + H] ⁺ ; HPLC 99.6% (AUC), T _R 6.52 min; UV (MeOH) λ _max 288 nm, ε 28,364.
Example 19
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (trans-4-((2-hydroxyethyl) carbamoyl) cyclohexyl) -4H-thieno [3,2-b] pyrrole -5- carboxamide (JRW-0043)

According to the general procedure B, trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane The 1-carboxylic acid (50 mg, 0.11 mmol) was reacted with ethanolamine (7 mg, 0.13 mmol) to give the desired product (48 mg, 88%) as a white foam. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.86 (d, J = 7.8, ¹ H), 7.69 (t, J = 5.4, ¹ H), 7.45-7. m, 5H), 7.13 to 7.04 (m, 2H), 4.96 (s, 2H), 4.60 (t, J = 5.5, 1H), 3.75 to 3.54 (m, 5H) m, 3H), 3.36 (q, J = 5.9, 2H), 3.08 (q, J = 5.9, 2H), 2.37 (s, 3H), 2.14-2. 00 (m, 1 H), 1.90-1.69 (m, 4 H), 1.52-1.10 (m, 4 H), 1.08-0.94 (m, 3 H); ESI MS m / ^{z 511 [M + H] +} ; HPLC99.7% (AUC), T R 6.52 _{min; UV (MeOH) λ max 289nm} , ε24,966.
Example 20
N- (trans-4-((2- (dimethylamino) ethyl) carbamoyl) cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2- b] Pyrrole-5-carboxamide (JRW-0044)

According to the general procedure B, trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane The 1-carboxylic acid (50 mg, 0.11 mmol) was reacted with ethanolamine (18 mg, 0.21 mmol) to give the desired product (45 mg, 78%) as a pale red solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.87 (d, J = 8. ^{1, 1} H), 7.62 (t, J = 5.5, ¹ H), 7.45-7. 35 ( m, 2H), 7.33-7.17 (m, 3H), 7.12-7.05 (m, 2H), 4.97 (s, 2H), 3.73-3.54 (m, 3H) 3H), 3.10 (q, J = 6.4, 2H), 2.37 (s, 3H), 2.24 (t, J = 6.4, 2H), 2.15-2.00 (2. m, 7H), 1.88-1.691.78 (m, 4H), 1.53-1.19 (m, 4H), 1.08-0.94 (m, 3H); ESI MS m / ^{z 538 [M + H] +} ; HPLC99.4% (AUC), T R 4.31 _{min; UV (MeOH) λ max 289nm} , ε29,980.
Example 21
4- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1-carboxamide) Butanoic acid (JRW-0051)
Step 1. 4- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1 -Carboxamide) methyl butanoate (JRW-0050)

According to the general procedure B, trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane The 1-carboxylic acid (50 mg, 0.11 mmol) was reacted with methyl 4-aminobutanoate (15 mg, 0.13 mmol) to give the crude product (80 mg) as a white glass. ESI MS m / z 567 [M + H] ^< +>.
Step 2. 4- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1 -Carboxamide) butanoic acid (JRW-0051)

4- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) according to General Procedure A )) Cyclohexane-1-carboxamido) Methyl butanoate (60 mg, 0.10 mmol) was reacted with lithium hydroxide (13 mg, 0.53 mmol) to give the desired product (55 mg, 93%) as a white solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 11.97 (s, 1 H), 7.82 (d, J = 7.7, ¹ H), 7.66 (t, J = 5.4, 1 H) , 7.40-7. 29 (m, 2 H), 7. 27-7. 13 (m, 3 H), 7.0 7-7.00 (m, 2 H), 4.9 2 (s, 2 H), 3 .68-3.48 (m, 3 H), 3.03-2.92 (m, 2 H), 2. 32 (s, 3 H), 2. 14 (t, J = 7.3, 2 H), 2 .06-1.93 (m, 1 H), 1.85-1.64 (m, 4 H), 1.62-1. 47 (m, 2 H), 1.47-1.15 (m, 4 H) , 1.05-0.88 (m, 3H); ESI MS m / z 553 [M + H] +; HPLC> 99% (AUC), T R 5.17 _{min; UV (MeOH) λ max 289nm} , ε24, 710.
Example 22
N- (trans-4-carbamoylcyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide (JRW-0052 )

According to the general procedure B, trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane The 1-carboxylic acid (50 mg, 0.11 mmol) was reacted with ammonia (1.1 mL, 0.5 M, 0.21 mmol) to give the desired product (45 mg, 90%) as a white solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.82 (d, J = 8.2, 1 H), 7.40-7.29 (m, 2 H), 7.28-7.08 (m, m) 4H), 7.06 to 6.98 (m, 2H), 6.61 (s, 1H), 4.91 (s, 2H), 3.67-3.49 (m, 3H), 2.31 (S, 3H), 2.03-1.91 (m, 1H), 1.82-1.68 (m, 4H), 1.47-1.14 (m, 4H), 1.03-0 .88 (s, 3H); ESI MS m / z 467 [M + H] +; HPLC> 99% (AUC), T R 5.11 _{min; UV (MeOH) λ max 289nm} , ε25,213.
Example 23
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (trans-4- (hexylcarbamoyl) cyclohexyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide ( JRW-0138)

According to the general procedure B, trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane The 1-carboxylic acid (50 mg, 0.11 mmol) was reacted with hexylamine (13 mg, 0.13 mmol) to give the desired product (50 mg, 85%) as a white solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.88 (d, J = 8. ^{1, 1} H), 7.67 (t, J = 5.6, ¹ H), 7.45-7.34 (7 m, 2H), 7.33-7.18 (m, 3H), 7.12-7.04 (m, 2H), 4.96 (s, 2H), 3.72-3.52 (m, 2H) 3H), 2.99 (dd, J = 6.5, 12.6, 2H), 2.36 (s, 3H), 2.10-1.96 (m, 1H), 1.88-1. 68 (m, 4 H), 1.52-1.16 (m, 12 H), 1.08-0.92 (m, 3 H), 0.88-0.78 (m, 3 H); ESI MS m / ^{z 551 [M + H] +} ; HPLC99.4% (AUC), T R 6.54 _{min; UV (EtOH) λ max 292nm} , ε29,535.
Example 24
1- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carbonyl) Ethyl piperidine-4-carboxylate (JRW-0140)

According to the general procedure B, trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane The 1-carboxylic acid (50 mg, 0.11 mmol) was reacted with ethyl piperidine-4-carboxylate (20 mg, 0.13 mmol) to give the desired product (60 mg, 92%) as a clear semisolid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.92 (d, J = 7.8, ¹ H), 7.45-7.34 (m, 2 H), 7.32-7.18 (m, 7) 3H), 7.12-7.05 (m, 2H), 4.96 (s, 2H), 4.23 (d, J = 12.7, 1H), 4.05 (q, J = 7. 1, 2 H), 3.87 (d, J = 12.7, 1 H), 3.72-3.52 (m, 3 H), 3.18-3.02 (m, 1 H), 2.76- 2.51 (m, 3 H), 2.36 (s, 3 H), 1.93 to 1.63 (m, 6 H), 1.56 to 1.26 (m, 6 H), 1.16 (t, 6) J = 6.0Hz, 3H), 1.07-0.93 (m, 3H); ESI MS m / z 607 [M + H] +; HPLC99.5% (AUC), T R 6.01 min; UV ( EtOH) λ _max 290 n m, ε 29, 325.
Example 25
6- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxamide) Methyl Hexanoate (JRW-0145)

According to the general procedure B, trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane The 1-carboxylic acid (50 mg, 0.11 mmol) was reacted with methyl 6-aminohexanoate (23 mg, 0.13 mmol) to give the desired product (60 mg, 94%) as a white solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.88 (d, J = 8. ^{1, 1} H), 7.68 (t, J = 5.6, ¹ H), 7.44-7. m, 2H), 7.32-7.17 (m, 3H), 7.12-7.03 (m, 2H), 4.95 (s, 2H), 3.73-3.50 (m, 3H) 6H), 2.98 (dd, J = 6.6, 12.5, 2H), 2.36 (s, 3H), 2.26 (t, J = 7.4, 2H), 2.08- 1.95 (m, 1 H), 1.87-1.67 (m, 4 H), 1.57-1. 15 (m, 10 H), 1.05-0.93 (m, 3 H); ESI MS ^{m / z 595 [M + H} ] +; HPLC99.4% (AUC), T R 6.24 _{min; UV (EtOH) λ max 288nm} , ε26,555.
Example 26
6- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxamide) Hexanoic acid (JRW-0147)

Following the general procedure A, 6- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide )) Cyclohexane-1-carboxamido) Methyl hexanoate (53 mg, 0.089 mmol) was reacted with lithium hydroxide (10 mg, 0.44 mmol) to give the desired product (50 mg, 96%) as a white solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 11.98 (s, 1 H), 7.88 (d, J = 7.6, 1 H), 7.72-7.63 (m, 1 H), 7 .44-7.33 (m, 2H), 7.32-7.18 (m, 3H), 7.12-7.04 (m, 2H), 4.95 (s, 2H), 3.72 −3.52 (m, 3 H), 3.04-2.93 (m, 2 H), 2.36 (s, 3 H), 2.16 (t, J = 7.1, 2 H), 2.10 -1.93 (m, 1 H), 1.88-1.67 (m, 4 H), 1.55-1.10 (m, 10 H), 1.08-0.92 (m, 3 H); ESI ^{MS m / z 581 [M +} H] +; HPLC> 99% (AUC), T R 5.49 _{min; UV (EtOH) λ max 288nm} , ε23,738.
Example 27
1- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carbonyl) Piperidine-4-carboxylic acid (JRW-0187)

Following the general procedure A, 1- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide ) Ethyl cyclohexane-1-carbonyl) piperidine-4-carboxylate (60 mg, 0.099 mmol) is reacted with lithium hydroxide (12 mg, 0.49 mmol) to give the desired product as a white solid (45 mg, 78%) I got ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 12.20 (s, 1 H), 7.90 (d, J = 7.5, 1 H), 7.45-7.33 (m, 2 H), 7 .32-7.18 (m, 3H), 7.08 (s, 2H), 4.96 (s, 2H), 4.28-4.15 (m, 1H), 3.92-3.79 (M, 1 H), 3.70-3.52 (m, 3 H), 3.15-3.00 (m, 1 H), 2.75-2. 60 (m, 1 H), 2.36 (s) , 3H), 1.92-1.63 (m, 7H), 1.56-1.25 (m, 7H), 1.08-0.95 (m, 3H); ESI MS m / z 579 [ ^{M + H] +; HPLC99.5%} (AUC), T R 4.45 _{min; UV (EtOH) λ max 289nm} , ε23,470.
Example 28
8- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxamide) Octanoic acid (JRW-0188)
Step 1.8- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1 -Carboxamido) methyl octanoate (JRW-0186)

According to the general procedure B, trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane The 1-carboxylic acid (50 mg, 0.11 mmol) was reacted with methyl 8-aminooctanoate (27 mg, 0.13 mmol) to give the desired product (55 mg, 82%) as an oil. ESI MS m / z 623 [M + H] ^< +>.
Step 2.8- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1 -Carboxamide) Octanoic acid (JRW-0188)

8- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) according to General Procedure A ) Cyclohexane-1-carboxamido) Methyl octanoate (50 mg, 0.080 mmol) was reacted with lithium hydroxide (9 mg, 0.40 mmol) to give the desired product (45 mg, 92%) as a white foam. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 11.92 (s, 1 H), 7.87 (d, J = 7.8, 1 H), 7.70-7.61 (m, 1 H), 7 .45-7.14 (m, 5H), 7.10-7.03 (m, 2H), 4.96 (s, 2H), 3.72-3.54 (m, 3H), 3.05 -2.93 (m, 2 H), 2.37 (s, 3 H), 2.22-2.12 (m, 2 H), 2. 10-1. 98 (m, 1 H), 1.9 1-1 .66 (m, 4 H), 1.55-1. 10 (m, 14 H), 1.08-0.93 (m, 3 H); ESI MS m / z 609 [M + H] ⁺ ; HPLC 96.9% ( AUC), T _R 4.97 _{min; UV (EtOH) λ max 289nm} , ε25,824.
Example 29
N- (trans-4- (cyclohexylcarbamoyl) cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide ( JRW-0190)

According to the general procedure B, trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane The 1-carboxylic acid (50 mg, 0.11 mmol) was reacted with cyclohexylamine (16 mg, 0.16 mmol) to give the desired product (55 mg, 94%) as a white solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.95 to 7.82 (m, 1 H), 7.66 to 7.16 (m, 6 H), 7.12 to 7.04 (m, 2 H) , 4.97 (s, 2 H), 3.76-3. 40 (m, 4 H), 2.36 (s, 3 H), 2.11-1. 96 (m, 1 H), 1.90-1 .60 (m, 8H), 1.59-0.91 (m, 13H); ESI MS m / z 549 [M + H] +; HPLC99.5% (AUC), T R 5.98 min; UV (EtOH ) Λ _max 288 nm, ε 25, 407.
Example 30
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (trans-4-((1-methylpiperidin-4-yl) carbamoyl) cyclohexyl) -4H-thieno [3,2 -B] pyrrole-5-carboxamide (JRW-0191)

According to the general procedure B, trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane The 1-carboxylic acid (50 mg, 0.11 mmol) was reacted with 1-methylpiperidin-4-amine (18 mg, 0.16 mmol) to give the desired product (47 mg, 78%) as a white solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.87 (d, J = 8. ^{1, 1} H), 7.58 (d, J = 7.6, ¹ H), 7. 50-7. 16 ( m, 5H), 7.13 to 7.04 (m, 2H), 4.96 (s, 2H), 3.75 to 3.38 (m, 4H), 2.73 to 2.61 (m, 5H) 2H), 2.36 (s, 3H), 2.22-1.96 (m, 5H), 1.96-1.58 (m, 8H), 1.53-1.19 (m, 6H) , 1.08-0.97 (s, 3H); ESI MS m / z 564 [M + H] +; HPLC98.8% (AUC), T R 3.35 _{min; UV (EtOH) λ max 289nm} , ε30, 051.
Example 31
4- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1-carboxamide) Piperidine-1-carboxylic acid tert-butyl (JRW-0192)

According to the general procedure B, trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane The 1-carboxylic acid (84 mg, 0.18 mmol) is reacted with tert-butyl 4-aminopiperidine-1-carboxylate (54 mg, 0.27 mmol) to give the desired product (115 mg, 99%) as a white solid Obtained. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.93 to 7.83 (m, 1 H), 7.68 to 7.58 (m, 1 H), 7.45 to 7.16 (m, 5 H) , 7.13-7.03 (m, 2 H), 4.96 (s, 2 H), 3.96-3.46 (m, 6 H), 2.93-2. 70 (m, 2 H), 2 .36 (s, 3 H), 2.09-1.95 (m, 1 H), 1.88-1.60 (m, 6 H), 1.55 to 1.08 (m, 15 H), 1.06 -0.93 (m, 3H); ESI MS m / z 650 [M + H] +; HPLC99.6% (AUC), T R 5.87 _{min; UV (EtOH) λ max 288nm} , ε31,128.
Example 32
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (trans-4- (piperidin-4-ylcarbamoyl) cyclohexyl) -4H-thieno [3,2-b] pyrrole- 5-carboxamide (JRW-0194)

4- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) in dichloromethane (5 mL) To a solution of cyclohexane-1-carboxamido) piperidine-1-carboxylate tert-butyl (105 mg, 0.16 mmol) was added trifluoroacetic acid (1 mL). The reaction was stirred at room temperature for 2 hours. The mixture was diluted with toluene and concentrated in vacuo (3 ×) to give a white foam crude product (150 mg). ESI MS m / z 550 [M + H] ^< +>.
Example 33
N- (trans-4-((1-acetylpiperidin-4-yl) carbamoyl) cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2 -B] pyrrole-5-carboxamide (JRW-0195)

4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (trans-4- (piperidin-4-ylcarbamoyl) cyclohexyl) -4H in dichloromethane (5 mL) cooled to 0 ° C. To a solution of -thieno [3,2-b] pyrrole-5-carboxamide (0.16 mmol) was added acetyl chloride (25 mg, 0.32 mmol) and diisopropylethylamine (104 mg, 0.81 mmol). The reaction was stirred and allowed to warm to room temperature overnight. The mixture was diluted with dichloromethane and water and the layers separated. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue was purified by column chromatography (silica, dichloromethane / methanol) to give the desired product as a white solid (84 mg, 88%). ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.87 (d, J = 7.6, ¹ H), 7.67 (d, J = 7.6, ¹ H), 7.46-7.32 ( m, 2H), 7.33-7.17 (m, 3H), 7.13-7.03 (m, 2H), 4.96 (s, 2H), 4.22-4.08 (m, 2H) 1H), 3.81 to 3.52 (m, 5H), 3.15 to 3.00 (m, 1H), 2.70 (t, J = 11.9, 1H), 2.36 (s, 2) 3H), 2.11-1.94 (m, 4H), 1.91-1.59 (m, 6H), 1.52-1.09 (m, 6H), 1.06-0.91 (m. m, 3H); ESI MS m / z 592 [M + H] +; HPLC99.8% (AUC), T R 4.09 _{min; UV (EtOH) λ max 288nm} , ε26,034.
Example 34
(6- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1-carboxamide ) Hexyl) tert-butyl carbamate (JRW-0197)

According to the general procedure B, trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane Reaction of 1-carboxylic acid (115 mg, 0.25 mmol) with tert-butyl (6-aminohexyl) carbamate (80 mg, 0.37 mmol) gives the desired product (150 mg, 91%) as a white foam The ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.87 (d, J = 8.0, ¹ H), 7.65 (t, J = 5.1, ¹ H), 7.45-7.33 ( m, 2H), 7.32-7.17 (m, 3H), 7.12-7.06 (m, 2H), 6.77-6.68 (m, 1H), 4.96 (s, 6) 2H), 3.72-3.53 (m, 3H), 3.03-2.93 (m, 2H), 2.87 (dd, J = 6.0, 12.6, 2H), 2. 36 (s, 3 H), 2.21 to 1.96 (m, 1 H), 1.88 to 1.68 (m, 4 H), 1.51 to 1.16 (m, 21 H), 1.06 to 0.93 (m, 3H); ESI MS m / z 666 [M + H] +; HPLC99.7% (AUC), T R 4.11 _{min; UV (EtOH) λ max 288nm} , ε21,608.
Example 35
N- (trans-4-((6- (3 ′, 6′-dihydroxy-3-oxo-3H-spiro [isobenzofuran-1,9′-xanthene] -5 (6) -carboxamido) hexyl) carbamoyl) Cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide (JRW-0200)
Step 1. N- (trans-4-((6-aminohexyl) carbamoyl) cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole -5- carboxamide (JRW-0199)

(6- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide in dichloromethane (5 mL) To a solution of tert-butyl cyclohexane-1-carboxamido) hexyl) carbamate (150 mg, 0.22 mmol) was added trifluoroacetic acid (1 mL). The reaction was stirred at room temperature for 5 hours. The mixture was diluted with toluene and concentrated (3 ×) in vacuo to give the crude product (150 mg) as a clear oil. ESI MS m / z 566 [M + H] ^< +>.
Step 2. N- (trans-4-((6- (3 ′, 6′-dihydroxy-3-oxo-3H-spiro [isobenzofuran-1,9′-xanthene] -5 (6) -carboxamido) hexyl) carbamoyl) Cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide (JRW-0200)

N- (trans-4-((6-aminohexyl) carbamoyl) cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3 in DMF (3 mL) 2, 2-dioxopyrrolidin-1-yl 3 ', 6'-dihydroxy-3-oxo-3H-spiro [isobenzofuran-1 in a solution of 2, 2-b] pyrrole-5-carboxamide (0.22 mmol) , 9'-xanthene] -5 (6) -carboxylic acid [5 (6) -FAM-SE] (106 mg, 0.22 mmol) and diisopropylethylamine (145 mg, 1.1 mmol) were added. The reaction was stirred at room temperature for 1 hour. The reaction mixture was acidified (0.1 M HCl), diluted with water and extracted with 3: 1 CHCl ₃ / isopropanol. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue was purified by column chromatography (silica, dichloromethane / methanol) to give the desired product (145 mg, 69%) as an orange solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 10.12 (s, 2 H), 8.78 (t, J = 5.5, ¹ H, isomer A), 8.64 (t, J = 5. 7, 1 H, Isomer B), 8.44 (s, 1 H, Isomer A), 8.23 (dd, J = 1.5, 8.1, 1 H, Isomer A), 8.15 (dd , J = 1.3, 8.1, 1 H, isomer B), 8.05 (d, J = 8.1, 1 H, isomer B), 7.87 (d, J = 8.0, 1 H) ), 7.72-7.60 (m, 1H), 7.44-7.32 (m, 3H), 7.32-7.17 (m, 3H), 7.12-7.04 (m) , 2H), 6.70-6.65 (m, 2H), 6.61-6.49 (m, 4H), 4.96 (s, 2H), 3.70-3.52 (m, 3H) ), 3.32-3.23 (m, 2H), 3.22-3. 2 (m, 1 H), 3.08-2.91 (m, 2 H), 2. 36 (s, 3 H), 2.11-1. 95 (m, 1 H), 1.89-1. 65 ( m, 4H), 1.61-1.16 (m, 11H), 1.06-0.92 (m, 3H); ESI MS m / z 925 [M + H] ⁺ ; HPLC 97.1% (AUC), T _R 6.16, 6.26 min; UV (MeOH) λ _max 284 nm, ε 31, 419.
Example 36
N- (trans-4-((6-aminohexyl) carbamoyl) cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole -5-carboxamide hydrochloride (JRW-0241)

(6- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide in dichloromethane (5 mL) To a solution of tert-butyl cyclohexane-1-carboxamido) hexyl) carbamate (100 mg, 0.15 mmol) was added trifluoroacetic acid (1 mL). The reaction was stirred at room temperature for 1 hour. The mixture was diluted with toluene and concentrated (3 ×) in vacuo. The residue was dissolved in methanol and HCl (2 mL, 1 M in ether) was added. The solution was evaporated to give the desired product as a white solid (95 mg, quant.). ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 8.02-7.68 (m, 5H), 7.45-7.34 (m, 2H), 7.33-7.15 (m, 3H) , 7.12-7.05 (m, 2H), 4.97 (s, 2H), 3.74-3.51 (m, 3H), 3.01 (dd, J = 6.5, 12. 5, 2 H), 2.79-2. 66 (m, 2 H), 2. 36 (s, 3 H), 2.1 1-1. 97 (m, 1 H), 1. 88-1. 68 (m, 2) 4H), 1.59-1.17 (m, 12H ), 1.02-0.94 (m, 3H); ESI MS m / z 566 [M + H] +; HPLC99.0% (AUC), T R 4.47 min; UV (MeOH) λ _max 289 nm, ε 23, 213.
Example 37
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (trans-4-((6-hydroxyhexyl) carbamoyl) cyclohexyl) -4H-thieno [3,2-b] pyrrole -5- carboxamide (JRW-0242)

According to the general procedure B, trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane 1- Carboxylic acid (150 mg, 0.32 mmol) was reacted with 6-aminohexan-1-ol (56 mg, 0.48 mmol) to give the desired product (160 mg, 88%) as a white foam. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.87 (d, J = 7.4, ¹ H), 7.73-7.59 (s, 1 H), 7.48-7.15 (m, 7) 5H), 7.13 to 7.02 (m, 2H), 4.96 (s, 2H), 4.35 to 4.23 (m, 1H), 3.73 to 3.50 (m, 3H) , 3.43-3.30 (m, 2H), 3.05-2.94 (m, 2H), 2.37 (s, 3H), 2.10-1.95 (m, 1H), 1 .90-1.65 (m, 4H), 1.55-1.13 (m, 12H), 1.09-0.90 (s, 3H); ESI MS m / z 567 [M + H] ⁺ ; HPLC > 99% (AUC), T R 5.48 _{min; UV (MeOH) λ max 289nm} , ε26,356.
Example 38
6- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxamide) Hexane-1-sulfonic acid sodium (JRW-0344)
Step 1. 4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (trans-4-((6-iodohexyl) carbamoyl) cyclohexyl) -4H-thieno [3,2- b] Pyrrole-5-carboxamide (JRW-0342)

4- (2- (ethyl (i)) dissolved in THF (5 mL) in a solution of imidazole (36 mg, 0.53 mmol), triphenylphosphine (138 mg, 0.53 mmol), and iodine (134 mg, 0.53 mmol) m-Tolyl) amino) -2-oxoethyl) -N- (trans-4-((6-hydroxyhexyl) carbamoyl) cyclohexyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide (100 mg, 0 .18 mmol) was added. The solution was stirred at room temperature for 1 hour. The reaction was diluted with ethyl acetate and quenched with 10% Na ₂ S ₂ O ₃ solution. The mixture was diluted with ethyl acetate and water and the layers separated. The organic layer was washed with 10% Na ₂ S ₂ O ₃ solution and brine, dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue was partially purified by column chromatography (silica, dichloromethane / methanol) to give the crude product as a white solid. ESI MS m / z 677 [M + H] ^< +>.
Step 2. 2- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1 -Carboxamide) Hexane-1-sulfonic acid sodium (JRW-0344)

4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (trans-4-((6-iodohexyl) carbamoyl) cyclohexyl) -4H-thieno [3 in ethanol (5 mL) To a solution of [2-b] pyrrole-5-carboxamide (0.18 mmol) was added sodium sulfite (66 mg, 0.53 mmol) and water (3 mL). The mixture was heated to 75 ° C. for 2 hours. The reaction is concentrated and the residue is purified by column chromatography (silica, dichloromethane / methanol) to give the desired product (100 mg, 90%) as a white solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.87 (d, J = 8.0, ¹ H), 7.67 (t, J = 5.5, ¹ H), 7.49-7. m, 5H), 7.13-7.05 (m, 2H), 4.97 (s, 2H), 3.72-3.54 (m, 3H), 3.05-2.93 (m, 5H) 2H), 2.40-2.28 (m, 5 H), 2.21-1.96 (m, 1 H), 1.88-1.68 (m, 4 H), 1.62-1.14 ( m, 12H), 1.05-0.93 (m , 3H); ESI MS m / z 631 [M + H] +; HPLC> 99% (AUC), T R 4.56 min; UV (MeOH) λ _max 288 nm, ε 21,072.
Example 39
6- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxamide) Hexane-1-sulfonic acid potassium (JRW-0348)

6- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) in water (25 mL) To a solution of cyclohexane-1-carboxamido) hexane-1-sulfonic acid (50 mg, 0.08 mmol) was added DowEx 50WX4 (charged potassium). The suspension was stirred at room temperature for 10 minutes. The mixture is filtered and the filtrate is concentrated by lyophilization to give the desired product (48 mg, 90%) as a white solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.83 (d, J = 8. ^{1, 1} H), 7.62 (t, J = 5.5, ¹ H), 7.47-7. m, 5H), 7.10-7.04 (m, 2H), 4.97 (s, 2H), 3.71-3.54 (m, 3H), 3.05-2.94 (m, 5H) 2H), 2.41 to 2. 31 (m, 5H), 2.15 to 1.96 (m, 1H), 1.90 to 1.70 (m, 4H), 1.61 to 1.16 (m. m, 12H), 1.06-0.95 (m , 3H); ESI MS m / z 631 [M + H] +; HPLC> 99% (AUC), T R 4.55 min; UV (MeOH) λ _max 289 nm, ε 19 812.
Example 40
Methyl-trans-4- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane 1-carboxamido) cyclohexane-1-carboxylic acid (JRW-0243)

According to the general procedure B, trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane The 1-carboxylic acid (75 mg, 0.16 mmol) is reacted with trans-4-aminocyclohexane-1-carboxylic acid methyl hydrochloride (46 mg, 0.24 mmol) to give the desired product (89 mg, 91%) as a white solid Got). ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.86 (d, J = 8.5, ¹ H), 7.58 (d, J = 7.8, ¹ H), 7.47-7.33 ( m, 2H), 7.32-7.18 (m, 3H), 7.11-7.04 (m, 2H), 4.96 (s, 2H), 3.72-3.53 (m, 2H) 6H), 3.52-3.35 (m, 1H), 2.36 (s, 3H), 2.29-2.16 (m, 1H), 2.08-1.95 (m, 1H) , 1.95 to 1.65 (m, 8 H), 1.53 to 1.06 (m, 8 H), 1.05 to 0.93 (m, 3 H); ESI MS m / z 607 [M + H] ⁺ ; HPLC99.7% (AUC), T R 6.27 _{min; UV (MeOH) λ max 288nm} , ε24,192.
Example 41
Trans-4- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1- Carboxamide) Cyclohexane-1-carboxylic acid (JRW-0245)

According to the general procedure A, methyl-trans-4- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole -5-carboxamido) cyclohexane-1-carboxamido) cyclohexane-1-carboxylic acid (80 mg, 0.13 mmol) is reacted with lithium hydroxide (16 mg, 0.66 mmol) to give the desired product (66 mg, as a white solid) 84%). ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 12.00 (s, 1 H), 7.86 (d, J = 8. ^{1, 1} H), 7.57 (d, J = 7.6, 1 H) , 7.45-7.33 (m, 2H), 7.32-7.15 (m, 3H), 7.12-7.03 (m, 2H), 4.97 (s, 2H), 3 .73-3.52 (m, 3 H), 3.52-3.35 (m, 1 H), 2.36 (s, 3 H), 2.19-1.95 (m, 2 H), 1.94 -1.65 (m, 8 H), 1.51-1.05 (m, 8 H), 1.05-0.94 (m, 3 H); ESI MS m / z 593 [M + H] ^< +>; HPLC 98.4 % (AUC), T _R 5.51 _{min; UV (MeOH) λ max 288nm} , ε24,976.
Example 42
(11S, 14S, 17S) -17-acetamido-11, 14-bis (carboxymethyl) -1- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl)- 4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexyl) -1,10,13,16-tetraoxo-2,9,12,15-tetraazanonadecane-19-oic acid (JRW-0251) )
Step 1. tert-Butyl (11S, 14S, 17S) -17-acetamido-11, 14-bis (2- (tert-butoxy) -2-oxoethyl) -1- (trans-4- (4- (2- (ethyl ( m-Tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexyl) -1,10,13,16-tetraoxo-2,9,12,15-tetra Azanona decane-19-oate (JRW-0249)

(S) -2-((S) -2-((S) -2-acetamido-4- (tert-butoxy) -4-oxobutanamido) -4- (tert-butoxy) according to the general procedure B ) -4-Oxobutanamido) -4- (tert-butoxy) -4-oxobutanoic acid (81 mg, 0.14 mmol) with N- (trans-4-((6-aminohexyl) carbamoyl) cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide hydrochloride (85 mg, 0.14 mmol) to give a pale yellow solid The desired product (125 mg, 78%) was obtained as ESI MS m / z 1122 [M + H] ^< +>.
Step 2. (11S, 14S, 17S) -17-acetamido-11, 14-bis (carboxymethyl) -1- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl)- 4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexyl) -1,10,13,16-tetraoxo-2,9,12,15-tetraazanonadecane-19-oic acid (JRW-0251) )

Tert-Butyl (11S, 14S, 17S) -17-acetamido-11, 14-bis (2- (tert-butoxy) -2-oxoethyl) -1- (trans-4- (4-) in dichloromethane (10 mL) (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexyl) -1,10,13,16-tetraoxo-2,9 To a solution of 12,15-tetraazanonadecane-19-oate (120 mg, 0.11 mmol) was added trifluoroacetic acid (1 mL). The reaction was stirred at room temperature for 18 hours. The mixture was diluted with toluene and concentrated (3 ×) in vacuo. The residue was purified by column chromatography (silica, dichloromethane / methanol) to give the desired product (68 mg, 66%) as a white solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 12.31 (s, 3 H), 8.45-8.18 (m, 2 H), 7.95-7.83 (m, 2 H), 7.66 (T, J = 5.5, 1 H), 7.52-7.17 (m, 6 H), 7.1 1-7.04 (m, 2 H), 4.96 (s, 2 H), 4.55 -4. 35 (m, 3 H), 3.71-3. 52 (m, 2 H), 3.06-2.90 (m, 4 H), 2.75-2.60 (m, 3 H), 2 .60-2.42 (m, 4 H), 2.36 (s, 3 H), 2.09-1.96 (m, 1 H), 1.89-1.66 (m, 7 H), 1.53 -1.13 (m, 12H), 1.06-0.93 (m, 3H); ESI MS m / z 953 [M + H] +; HPLC93.8% (AUC), T R 4.74 min; UV (MeOH) λ _{m ax} 289 nm, ε24, 417.
Example 43
Alternative synthesis of compounds of formula (Ia)

Compounds of formula (Ia) can be synthesized generally according to scheme 3.
Scheme 3.
Example 44
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N-methyl-4H-thieno [3,2-b] pyrrole-5-carboxamide (WZ-141-84)
Step 1. 3,5-Dioxopyrrolidin-1-yl 4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (WZ-141-82)

4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (0.44 g, 1.28 mmol) and TSTU (1. 16 g, 3.85 mol) were dissolved in 15 ml of methylene chloride and 15 ml of acetonitrile. DIPEA (0.996 g, 7.71 mmol) was added slowly at room temperature, and the resulting mixture was stirred at room temperature for 30 minutes. The reaction mixture was diluted with 100 ml of methylene chloride, washed twice with 30% citric acid, washed twice with water and dried over Na ₂ SO ₄ . The organic solvent was concentrated to a 30 ml solution. A portion of the solution was used directly in the next step without further purification.
Step 2. 4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N-methyl-4H-thieno [3,2-b] pyrrole-5-carboxamide (WZ-141-84)

10 ml of crude 2,5-dioxopyrrolidin-1-yl 4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5 above To carboxylic acid (WZ141-82) (150 mg, 0.34 mmol) methylamine (40%) (0.53 g, 6.83 mmol) was added and the resulting mixture was stirred at room temperature for 30 minutes. After removal of the solvent, the compound is purified by flash column using heptane / ethyl acetate as eluent to give the desired product in quantitative yield. ¹ H NMR (300 MHz, CD ₂ Cl ₂ ) δ 7.5-6.8 (m, 7 H), 4.95 (s, 2 H), 3.76 (m, 2 H), 2.91 (d, 3 H) ), 2.43 (s, 3 H), 1. 10 (t, 3 H); ESI MS m / z 356 [M + H] ⁺ ; 99.6% at HPLC 254 nm.
Example 45
N-Cyclopentyl-4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide (WZ-141-88)

The compound WZ141-88 is 4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N-methyl-4H-thieno [3,2-b] pyrrole-5-carboxamide (WZ141-84) Were synthesized using similar methods to prepare. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.96 (d, 1 H), 7.5-7.1 (m, 6 H), 6.8 (d, ¹ H), 4. 90 (s, 2 H) ), 4.61 (m, 1 H), 3.63 (m, 2 H), 2.38 (s, br, 3 H), 1.9-1.4 (m, 8 H), 1.00 (t, 3H); ESI MS m / z 410 [M + H] ⁺ ; HPLC purity 99.1% at 254 nm.
Example 46
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (pyridin-4-ylmethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide (WZ-141- 89)

The compound WZ141-89 is 4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N-methyl-4H-thieno [3,2-b] pyrrole-5-carboxamide (WZ141-84) Were synthesized using similar methods to prepare. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 8.79 (t, 1 H), 8.46 (d, 2 H), 7.45-7. 05 (m, 8 H), 4.92 (s, 2 H) ), 4.41 (d, 2 H), 3.58 (m, 2 H), 2. 32 (s, 3 H), 1. 10 (t, 3 H); ESI MS m / z 433 [M + H] ⁺ ; HPLC 94.2% at 254 nm purity.
Example 47
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (3-morpholinopropyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide (WZ-141- 90)

The compound WZ141-90 is 4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N-methyl-4H-thieno [3,2-b] pyrrole-5-carboxamide (WZ141-84) Were synthesized using similar methods to prepare. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 8.12 (d, 2 H), 7.4 to 7.0 (m, 6 H), 4.93 (s, 2 H), 3.7 to 3.4 (M, 6 H), 3.4 to 3.2 (m, 2 H), 2.4 to 2.2 (s, 9 H), 1.7 to 1.5 (m, 2 H), 1. 10 (t , 3H); ESI MS m / z 469 [M + H] ⁺ ; HPLC purity 98.0% at 254 nm.
Example 48
N-ethyl-2- (5- (4-methylpiperazine-1-carbonyl) -4H-thieno [3,2-b] pyrrol-4-yl) -N- (m-tolyl) acetamide (WZ-141-) 91)

The compound WZ141-91 is 4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N-methyl-4H-thieno [3,2-b] pyrrole-5-carboxamide (WZ141-84) Were synthesized using similar methods to prepare. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.44 (d, 1 H), 7.3-7.1 (m, 3 H), 7.03 (d, 1 H), 6.26 (s, 1 H) ), 4.68 (s, 2 H), 3.7-3.5 (m, 6 H), 2. 35 (s, 3 H), 2. 32 (m, 4 H), 2. 18 (s, 3 H) , 1.10 (t, 3 H); ESI MS m / z 425 [M + H] ⁺ ; HPLC purity 96.0% at 254 nm.
Example 49
Synthesis of compounds of formula (Ic)

Compounds of formula (Ic) can be generally synthesized according to Scheme 4.
Scheme 4.
Example 50
Methyl 4- (2-oxo-2- (m-tolylamino) ethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate (JRW-0077)

To a solution of 2-chloro-N- (m-tolyl) acetamide (500 mg, 2.7 mmol) in acetonitrile (20 mL), methyl 4H-thieno [3,2-b] pyrrole-5-carboxylate (411 mg, 2 .3 mmol), potassium carbonate (376 mg, 2.7 mmol) and 18-crown-6 (30 mg, 0.11 mmol) were added. The reaction was heated to reflux for 1.5 h. The mixture was diluted with ethyl acetate and water and the layers separated. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue was purified by column chromatography (silica, heptane / ethyl acetate) to give the desired product (530 mg, 71%) as a pale brown solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 10.21 (s, 1 H), 7.57 (d, J = 5.4, 1 H), 7.42 (s, 1 H), 7.37-7 .30 (m, 1 H), 7.27 (d, J = 5.4, 1 H), 7.23 (s, 1 H), 7.16 (t, J = 7.8, 1 H), 6.85 (D, J = 7.8, 1 H), 5.31 (s, 2 H), 3.73 (s, 3 H), 2. 24 (s, 3 H); ESI MS m / z 329 [M + H] ⁺ ; _{HPLC99.6% (AUC), T R} 6.32 _{min; UV (MeOH) λ max 288nm} , ε29,177.
Example 51
N-Cyclohexyl-4- (2-oxo-2- (m-tolylamino) ethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide (JRW-0081)
Step 1. 4- (2-Oxo-2- (m-tolylamino) ethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (JRW-0080)

Following general procedure A, methyl 4- (2-oxo-2- (m-tolylamino) ethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate (130 mg, 0.39 mmol) in water Reaction with lithium oxide (47 mg, 2.0 mmol) gave the crude product as a pale brown solid.
Step 2. N-Cyclohexyl-4- (2-oxo-2- (m-tolylamino) ethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide (JRW-0081)

Following general procedure B, 4- (2-oxo-2- (m-tolylamino) ethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (120 mg, 0.38 mmol) with cyclohexylamine Reaction with (56 mg, 0.57 mmol) gave the desired product (47 mg, 31%) as a white solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 10.13 (s, 1 H), 7.95 (d, J = 8. ^{1, 1} H), 7.43-7.38 (m, 2 H), 7 .33 (d, J = 8. 1, 1 H), 7.23-7. 10 (m, 3 H), 6. 84 (d, J = 7.5, 1 H), 5. 30 (s, 2 H) , 3.75 to 3.58 (m, 1 H), 2.24 (s, 3 H), 1.85 to 1.53 (m, 5 H), 1.35 to 1.00 (m, 5 H); MS m / z 396 [M + H] ^< +>; HPLC 94.7% (AUC), T _R 7.34 min; UV (MeOH) [lambda] _max 285 nm, [epsilon] 28, 066.
Example 52
N-Cyclohexyl-4- (2- (2- (2- (2-methoxyethoxy) ethyl) (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide ( JRW-0109)
Step 1. N- (2- (2-Methoxyethoxy) ethyl) -3-methylaniline (JRW-0101)

To a solution of m-toluidine (1.0 g, 9.3 mmol) in DMF (10 mL), 1-bromo-2- (2-methoxyethoxy) ethane (0.85 g, 4.6 mmol) and diisopropylamine (1. 2 g, 0.93 mmol) were added. The mixture was heated to 100 ° C. for 4 hours. The mixture was diluted with ethyl acetate and water and the layers separated. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue is purified by column chromatography (silica, heptane / ethyl acetate) to give the desired product (650 mg, 66%) as an oil. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.07 (t, J = 7.5, ¹ H), 6.56 (d, J = 7.5, ¹ H), 6.52-6.45 (m, 2H), 3.71 (t, J = 5.3, 2H), 3.67-3.62 (m, 2H), 3.60-3.52 (m, 2H), 3.31 (t, J = 5.3, 2H), 2.28 (s, 3H); ESI MS m / z 210 [M + H] ^< +>.
Step 2. 2-Chloro-N- (2- (2-methoxyethoxy) ethyl) -N- (m-tolyl) acetamide (JRW-0104)

To a solution of N- (2- (2-methoxyethoxy) ethyl) -3-methylaniline (650 mg, 3.1 mmol) in ethyl acetate (15 mL) was added water (5 mL). The biphasic solution was cooled to 0 ° C. and potassium hydroxide (522 mg, 9.3 mmol) was added in one portion. 2-Chloroacetyl chloride (526 mg, 4.7 mmol) was added dropwise over 10 minutes. The mixture was stirred for 2.5 hours, diluted with water and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated to give the crude product (830 mg) as a pale red oil. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.34-7.25 (m, 1 H), 7.18 (d, J = 7.7, ¹ H), 7.15-7.03 (m, 2 H) , 3.89 (t, J = 5.8, 2H), 3.83 (s, 2H), 3.65 (t, J = 5.8, 2H), 3.62-3.55 (m, 5) 2H), 3.54-3.46 (m, 2H), 2.37 (s, 3H); ESI MS m / z 286 [M + H] ^< +>.
Step 3.4- (2-((2- (2-Methoxyethoxy) ethyl) (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate methyl (JRW-0105)

In a solution of 2-chloro-N- (2- (2-methoxyethoxy) ethyl) -N- (m-tolyl) acetamide (830 mg, 2.9 mmol) in acetonitrile (20 mL) 4H-thieno [3,2 -B] Methyl pyrrole-5-carboxylate (438 mg, 2.4 mmol), potassium carbonate (400 mg, 2.9 mmol) and 18-crown-6 (32 mg, 0.12 mmol) were added. The mixture was heated to reflux for 18 hours. The mixture was diluted with ethyl acetate and water and the layers separated. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue was purified by column chromatography (silica, heptane / ethyl acetate) to give the crude product (1.3 g) as a thick oil. ESI MS m / z 430 [M + H] ^< +>.
Step 4. 4- (2-((2- (2-Methoxyethoxy) ethyl) (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid JRW-0107)

Following the general procedure A, 4- (2-((2- (2-methoxyethoxy) ethyl) (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5 Methyl carboxylate (2.4 mmol) was reacted with lithium hydroxide (287 mg, 12.0 mmol) to give the crude product (1.0 g, quant.) As a pale yellow solid. ESI MS m / z 417 [M + H] ^< +>.
Step 5. N-Cyclohexyl-4- (2- (2- (2- (2-methoxyethoxy) ethyl) (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide ( JRW-0109)

4- (2-((2- (2-methoxyethoxy) ethyl) (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5 according to General Procedure B -Carboxylic acid (120 mg, 0.28 mmol) was reacted with cyclohexylamine (42 mg, 0.43 mmol) to give the desired product (120 mg, 83%) as a white gum. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.96 to 7.85 (m, 1 H), 7.48 to 7.30 (m, 4 H), 7.30 to 7.20 (m, 1 H) , 7.17-7.07 (m, 2 H), 5.01 (s, 2 H), 3.82-3.62 (m, 3 H), 3.56-3.35 (m, 6 H), 3 .25 (s, 3 H), 2. 38 (s, 3 H), 1. 90-1.5 4 (m, 5 H), 1.4 1-1.0 4 (m, 5 H); ESI MS m / z 498 [ M + H] ⁺ ; HPLC 96.0% (AUC), T _R 7.38 min; UV (EtOH) λ _max 289 nm, ε 25, 434.
Example 53
Methyl-trans-4- (4- (2- (2- (2-methoxyethoxy) ethyl) (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5 -Carboxamide) cyclohexane 1-carboxylic acid (JRW-0110)

4- (2-((2- (2-methoxyethoxy) ethyl) (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5 according to General Procedure B -Carboxylic acid (120 mg, 0.28 mmol) is reacted with methyl trans-4-aminocyclohexane-1-carboxylate (68 mg, 0.43 mmol) to give the desired product (140 mg, 87%) as a white gum The ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.94 (d, J = 7.8, ¹ H), 7.46-7.20 (m, 5 H), 7.15-7.04 (m, 5) 2H), 5.00 (s, 2H), 3.78-3.65 (m, 3H), 3.60 (s, 3H), 3.52-3.35 (m, 6H), 3.23 (S, 3H), 2.38 (s, 3H), 2.34-2.20 (m, 1H), 2.02-1.81 (m, 4H), 1.53-1.22 (m , 4H); ESI MS m / z 556 [M + H] +; HPLC98.7% (AUC), T R 6.60 _{min; UV (MeOH) λ max 289nm} , ε23,567.
Example 54
4- (2- (Hexyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate methyl (JRW-0142)
Step 1. 2-Chloro-N-hexyl-N- (m-tolyl) acetamide (JRW-0141)

Water (5 mL) was added to a solution of N-hexyl-3-methylaniline (500 mg, 2.6 mmol) in ethyl acetate (15 mL). The biphasic solution was cooled to 0 ° C. and potassium hydroxide (440 mg, 7.8 mmol) was added in one portion. 2-Chloroacetyl chloride (442 mg, 3.9 mmol) was added dropwise over 10 minutes. The mixture was stirred for 1 hour, diluted with water and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated to give the crude product (730 mg) as a pale red oil. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.32 (t, J = 8.0, ¹ H), 7.24-7.17 (m, 1 H), 7.04-6.97 (m, 2 H) , 3.80 (s, 2 H), 3.74-3.61 (m, 2 H), 2.38 (s, 3 H), 1.59-1.41 (m, 2 H), 1.37-1 ESI MS m / z 268 [M + H] ^< +> .16 (m, 6 H), 0.92-0.80 (m, 3 H).
Step 2. 2- (2- (Hexyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate methyl (JRW-0142)

Methyl 4-H-thieno [3,2-b] pyrrole-5-carboxylate in a solution of 2-chloro-N-hexyl-N- (m-tolyl) acetamide (700 mg, 2.6 mmol) in acetonitrile (20 mL) (394 mg, 2.2 mmol), potassium carbonate (361 mg, 2.6 mmol) and 18-crown-6 (29 mg, 0.11 mmol) were added. The mixture was heated to reflux for 18 hours. The mixture was diluted with ethyl acetate and water and the layers separated. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated. The residue is purified by column chromatography (silica, heptane / ethyl acetate) to give the desired product (780 mg, 86%) as a white solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.53 (d, J = 5.4, ¹ H), 7.41 (t, J = 7.7, ¹ H), 7.33-7.11 ( m, 5H), 4.93 (s, 2H), 3.74 (s, 3H), 3.58 (t, J = 6.0 Hz, 2H), 2.37 (s, 3H), 1.48 −1.30 (m, 2H), 1.28−1.15 (m, 6H), 0.81 (t, J = 6.7, 3H); ESI MS m / z 413 [M + H] ⁺ ; HPLC 97 % (AUC), T _R 7.18 min; UV (EtOH) λ _max 289 nm, ε 26, 840.
Example 55
N-Cyclohexyl-4- (2- (hexyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide (JRW-0148)
Step 1. 4- (2- (Hexyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (JRW-0149)

Following the general procedure A, methyl 4- (2- (hexyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate (300 mg, 0.73 mmol ) Was reacted with lithium hydroxide (87 mg, 3.6 mmol) to give the crude product (300 mg) as a pale yellow solid. ESI MS m / z 399 [M + H] ^< +>.
Step 2. N-Cyclohexyl-4- (2- (hexyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide (JRW-0148)

4- (2- (Hexyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (50 mg, 0.12 mmol) according to General Procedure B Was reacted with cyclohexylamine (15 mg, 0.15 mmol) to give the desired product (60 mg, 99%) as a white foam. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.85 (d, J = 8.2, 1 H), 7.46 to 7.16 (m, 5 H), 7.12 to 7.06 (m, 5) 2H), 4.96 (s, 2H), 3.75-3.60 (m, 1H), 3.56 (t, J = 6.7, 2H), 2.36 (s, 3H), 1 .84-1.52 (m, 5H), 1.43-1.04 (m, 13H), 0.81 (t, J = 6.9, 3H); ESI MS m / z 480 [M + H] ⁺ ; HPLC98.1% (AUC), T R 8.62 _{min; UV (EtOH) λ max 288nm} , ε24,544.
Example 56
Methyl-trans-4- (4- (2- (hexyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1-carboxylic acid JRW-0149)

4- (2- (Hexyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (50 mg, 0.12 mmol) according to General Procedure B Was reacted with methyl trans-4-aminocyclohexane-1-carboxylate (24 mg, 0.15 mmol) to give the desired product (65 mg, 96%) as a white foam. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.91 (d, J = 8.0, ¹ H), 7.44-7.33 (m, 2 H), 7.33-7.17 (m, 5) 3H), 7.12-7.06 (m, 2H), 4.96 (s, 2H), 3.75-3.47 (m, 6H), 2.36 (s, 3H), 2.31 -2.20 (m, 1 H), 1.99-1. 78 (m, 4 H), 1.51-1. 10 (m, 12 H), 0.81 (t, J = 6.9, 3 H) ; ESI MS m / z 538 [ M + H] +; HPLC99.8% (AUC), T R 8.17 _{min; UV (EtOH) λ max 289nm} , ε26,509.
Example 57
Trans-4- (4- (2- (Hexyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid (JRW- 0260)

According to the general procedure A, methyl-trans-4- (4- (2- (hexyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) Cyclohexane-1-carboxylic acid (58 mg, 0.11 mmol) was reacted with lithium hydroxide (13 mg, 0.54 mmol) to give the desired product (55 mg, 97%) as a white foam. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 12.03 (s, 1 H), 7.88 (d, J = 7.8, ¹ H), 7.46-7.17 (m, 5 H), 7 .12-7.06 (m, 2 H), 4.97 (s, 2 H), 3.74-3.48 (m, 3 H), 2.36 (s, 3 H), 2.19-2.05 (M, 1 H), 2.00-1. 77 (m, 4 H), 1. 48-1.09 (m, 12 H), 0.88-0.77 (m, 3 H); ESI MS m / z ^{524 [M + H] +;} HPLC> 99% (AUC), T R 7.42 _{min; UV (EtOH) λ max 288nm} , ε24,240.
Example 58
Methyl 4- (2- (benzyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate (WZ-141-86)
Step 1. N-benzyl-2-chloro-N- (m-tolyl) acetamide (WZ-141-85)

To a solution of N-benzyl-3-methylaniline (1.35 g, 6.84 mmol) and TEA (0.761 g, 7.53 mmol) in 50 ml of methylene chloride was added 2-chloroacetyl chloride (0. 0) at 0 ° C. 772 g, 0.772 mmol) was slowly added. The resulting mixture was stirred at 0 ° C. for 30 minutes and then at room temperature overnight. The mixture was diluted with 100 ml of methylene chloride, washed three times with water and the organic layer was dried over Na ₂ SO ₄ . After removal of the solvent, the compound was purified by flash column chromatography using heptane and ethyl acetate as solvent to give a yellowish product in 74% yield. ¹ H NMR (300 MHz, CD ₂ Cl ₂ ) δ 7.6-6.7 (m, 9 H), 4.89 (s, 2 H), 3.71 (s, 2 H), 2.31 (s, 3 H) ); ESI MS m / z 274 [M + H] ^< +>.
Step 2. 2- (2- (Benzyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate methyl (WZ-141-86)

N-benzyl-2-chloro-N- (m-tolyl) acetamide (1.36 g, 4.97 mmol) in 50 ml acetonitrile, methyl 4H-thieno [3,2-b] pyrrole-5-carboxylate (0 .6g, 3.31mmol), 18- crown-6 ether (0.262g, 0.993mmol) and K ₂ CO ₃ (0.915g, a mixture of 6.62 mmol) was heated to reflux overnight. On cooling, most of the solvent was removed under vacuum and the residue was dissolved in methylene chloride (100 ml) and washed with water. The organic layer was dried over Na ₂ SO ₄ . After removal of the solvent, the compound was purified by flash column using heptane and ethyl acetate as solvent to give a pale product in 95% yield. ¹ H NMR (300 MHz, DMSO-d6) δ 7.57 (s, 1 H), 7.4 to 7.1 (m, 11 H), 5.06 (s, 2 H), 4.83 (s, 2 H) , 3.79 (s, 3 H), 2.31 (s, 3 H); ESI MS m / z 419 [M + H] ⁺ ; HPLC purity 94.3% at 254 nm.
Scheme 5.
Example 59
Trans-4- (4- (2- (ethyl (phenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) methyl cyclohexane-1-carboxylate (JRW-0318 )
Step 1. Methyl 4- (2- (tert-butoxy) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate (JRW-0277)

Potassium carbonate (1.28 g, 9.28 mmol), a solution of methyl 4H-thieno [3,2-b] pyrrole-5-carboxylate (1.4 g, 7.73 mmol) in acetonitrile (30 mL), 18- Crown-6 ether (102 mg, 0.38 mmol) and tert-butyl 2-bromoacetate (1.81 g, 9.28 mmol) were added. The suspension was heated to 75 ° C. for 18 hours. The mixture was diluted with ethyl acetate and water and the layers separated. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue was purified by column chromatography (silica, heptane / ethyl acetate) to give the desired product (2.1 g, 91%) as a pale yellow solid. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.34 (d, J = 5.4, ¹ H), 7.22 (d, J = 0.7, ¹ H), 6.87 (dd, J = 0) 7, 5.4, 1 H), 5.11 (s, 2 H), 3. 84 (s, 3 H), 1.46 (s, 9 H); ESI MS m / z 296 [M + H] ^< +>.
Step 2. 2- (5- (Methoxycarbonyl) -4H-thieno [3,2-b] pyrrol-4-yl) acetic acid (JRW-0288)

A solution of methyl 4- (2- (tert-butoxy) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate (1.92 g, 6.50 mmol) in DCM (20 mL) To this was added trifluoroacetic acid (2 mL). After the solution was stirred at room temperature for 4 hours, the mixture was concentrated. Toluene was added to the residue and concentrated to give the crude product as a white solid. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.38 (d, J = 5.4, ¹ H), 7.24 (s, 1 H), 6.89 (d, J = 5.4, 1 H), 5 .24 (s, 2 H), 3.86 (s, 3 H); ESI MS m / z 240 [M + H] ^< +>.
Step 3.4- (2- (Ethyl (phenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate methyl (JRW-0294)

To a solution of 2- (5- (methoxycarbonyl) -4H-thieno [3,2-b] pyrrol-4-yl) acetic acid (100 mg, 0.42 mmol) in DMF (3 mL) was added N-ethylaniline (101 mg) , 0.84 mmol), HATU (318 mg, 0.84 mmol), and diisopropylethylamine (108 mg, 0.84 mmol) were added. The reaction was heated to 75 ° C. for 18 hours. The reaction mixture was cooled, diluted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue was purified by column chromatography (silica, heptane / ethyl acetate) to give the desired product (86 mg, 60%) as a white solid. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.57 to 7.21 (m, 6 H), 7.16 (s, 1 H), 6.80 (d, J = 5.5, 1 H), 4.95 (S, 2 H), 3.87-3. 68 (m, 5 H), 1. 13 (t, J = 7.2, 3 H); ESI MS m / z 343 [M + H] ^< +>.
Step 4. 4- (2- (Ethyl (phenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (JRW-309)

Following the general procedure A, methyl 4- (2- (ethyl (phenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate (86 mg, 0.25 mmol) Reaction with lithium hydroxide (30 mg, 1.3 mmol) gave the crude product (82 mg) as a pale brown solid. ESI MS m / z 329 [M + H] ^< +>.
Step 5. Trans-4- (4- (2- (ethyl (phenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) methyl cyclohexane-1-carboxylate (JRW-0318 )

According to General Procedure B, trans 4- (2- (ethyl (phenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (80 mg, 0.24 mmol) in trans Reaction with methyl -4-aminocyclohexane-1-carboxylate (56 mg, 0.29 mmol) gave the desired product (81 mg, 71%) as a white solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.89 (d, J = 7.9, ¹ H), 7.60-7.28 (m, 6 H), 7.04-7.13 (m, 6) 2H), 4.95 (s, 2H), 3.73-3.52 (m, 5H), 2.31-2.18 (m, 1H), 2.03-1.78 (m, 4H) , 1.50-1.20 (m, 4H), 1.07-0.93 (s, 3H); ESI MS m / z 468 [M + H] +; HPLC97.7% (AUC), T R 6. 51 minutes; UV (EtOH) λ _max 289 nm, ε 28, 274.
Example 60
Trans-4- (4- (2-((3-Cyanophenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid Methyl (JRW-0321)
Step 1. 2-Chloro-N- (3-cyanophenyl) -N-ethylacetamide (JRW-0313)

Water (3 mL) was added to a solution of 3- (ethylamino) benzonitrile (60 mg, 0.41 mmol) in ethyl acetate (7 mL). The biphasic solution was cooled to 0 ° C. and potassium hydroxide (69 mg, 1.2 mmol) was added in one portion. 2-Chloroacetyl chloride (69 mg, 0.62 mmol) was added dropwise over 10 minutes. The mixture was stirred for 2 hours, diluted with water and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated to give the crude product (98 mg) as an oil. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.81 to 7.42 (m, 4 H), 3.84 to 3.70 (m, 4 H), 1.15 (t, J = 7.2, 3 H) ESI MS m / z 223 [M + H] ⁺ .
Step 2. 2- (2-((3-Cyanophenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate methyl (JRW-0316)

Potassium carbonate (62 mg, 0.45 mmol), 18-crown-6 ether in a solution of methyl 4H-thieno [3,2-b] pyrrole-5-carboxylate (68 mg, 0.37 mmol) in acetonitrile (5 mL) (5 mg, 0.019 mmol) and 2-chloro-N- (3-cyanophenyl) -N-ethylacetamide (98 mg, 0.45 mmol) were added. The suspension was heated to 75 ° C. for 18 hours. The mixture was diluted with ethyl acetate and water and the layers separated. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue is purified by column chromatography (silica, heptane / ethyl acetate) to give the desired product (94 mg, 68%) as a white foam. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.71-7.62 (m, 1 H), 7.61-7.49 (m, 3 H), 7.33 (d, J = 5.4, ¹ H) , 7.12 (s, 1 H), 6.82 (d, J = 5.4, 1 H), 4.98 (s, 2 H), 3.87-3.70 (m, 5 H), 1.14 (T, J = 7.1, 3 H); ESI MS m / z 368 [M + H] ^< +>.
Step 3. 4- (2-((3-cyanophenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (JRW-0319-1) and 4- (2-((3-Carbamoylphenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (JRW-0319-2)

According to the general procedure A, methyl 4- (2-((3-cyanophenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate (90 mg, 0.24 mmol) was reacted with lithium hydroxide (12 mg, 0.49 mmol). The two products were separated by column chromatography (silica, dichloromethane / methanol) to give the nitrile (61 mg) and the amide (31 mg) as a white solid. ESI MS m / z 354 [M + H] ^< +> and m / z 372 [M + H] ^< +>.
Step 4. Trans-4- (4- (2-((3-Cyanophenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid Methyl (JRW-0321)

According to the general procedure B, 4- (2-((3-cyanophenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (60 mg, 0 .17 mmol) was reacted with methyl trans-4-aminocyclohexane-1-carboxylate (56 mg, 0.29 mmol) to give the desired product (68 mg, 81%) as a white foam. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 8.02-7.59 (m, 5 H), 7.38 (d, J = 5.3, 1 H), 7.17-7.03 (m, 5) 2H), 5.02 (s, 2H), 3.77-3.55 (m, 6H), 2.32-2.18 (m, 1H), 1.99-1.79 (m, 4H) , 1.54-1.20 (m, 4H), 1.10-0.95 (m, 3H); ESI MS m / z 493 [M + H] +; HPLC98.3% (AUC), T R 6. 12 minutes; UV (EtOH) λ _max 289 nm, ε 26, 802.
Example 61
Trans-4- (4- (2-((3-carbamoylphenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid Methyl (JRW-0322)

According to the general procedure B, 4- (2-((3-carbamoylphenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (30 mg, 0 .08 mmol) was reacted with methyl trans-4-aminocyclohexane-1-carboxylate (19 mg, 0.10 mmol) to give the desired product (33 mg, 80%) as a white foam. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 8.05 (s, 1 H), 7.97-7.83 (m, 3 H), 7.72-7.43 (m, 3 H), 7.37 (D, J = 5.4, 1 H), 7.12-7.05 (m, 2 H), 4.98 (s, 2 H), 3.75-3.54 (m, 6 H), 2. 32 -2.18 (m, 1 H), 1.99-1.79 (m, 4 H), 1.53-1.22 (m, 4 H), 1.10-0.95 (m, 3 H); ^{MS m / z 511 [M +} H] +; HPLC98.2% (AUC), T R 4.81 _{min; UV (EtOH) λ max 289nm} , ε28,223.
Example 62
Trans 4- (4- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) methyl cyclohexane-1-carboxylate JRW-0326)
Step 1. Methyl 4- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate (JRW-0298)

N, 3-diethylaniline in a solution of 2- (5- (methoxycarbonyl) -4H-thieno [3,2-b] pyrrol-4-yl) acetic acid (100 mg, 0.42 mmol) in DMF (3 mL) (94 mg, 0.63 mmol), HATU (318 mg, 0.84 mmol), and diisopropylethylamine (162 mg, 1.25 mmol) were added. The reaction was heated to 85 ° C. for 18 hours. The reaction mixture was cooled, diluted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue is purified by column chromatography (silica, heptane / ethyl acetate) to give the desired product (107 mg, 69%) as an orange foam. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.38 (t, J = 7.9, ¹ H), 7.31-7.20 (m, 2 H), 7.17-7.10 (m, 3 H) , 6.81 (d, J = 5.4, 1 H), 4.98 (s, 2 H), 3.84-3.67 (m, 5 H), 2.71 (q, J = 7.6, 2H), 1.29 (t, J = 7.6, 3H), 1.13 (t, J = 7.2, 3H); ESI MS m / z 371 [M + H] ⁺ .
Step 2. 2- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (JRW- 0323)

Following the general procedure A, methyl 4- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate (100 mg, 0. 1). 27 mmol) was reacted with lithium hydroxide (19 mg, 0.81 mmol) to give the crude product (100 mg) as a pale yellow solid. ESI MS m / z 357 [M + H] ^< +>.
Step 3. Trans 4- (4- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) methyl cyclohexane-1-carboxylate JRW-0326)

4- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (100 mg, 0.28 mmol) according to General Procedure B ) Was reacted with methyl trans-4-aminocyclohexane-1-carboxylate (65 mg, 0.34 mmol) to give the desired product (105 mg, 75%) as a white foam. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.90 (d, J = 7.9, ¹ H), 7.51-7.19 (m, 5 H), 7.13-7.04 (m, 5) 2H), 4.95 (s, 2H), 3.74-3.54 (m, 5H), 2.73-2.61 (m, 2H), 2.32-2.18 (m, 1H) , 2.01-1.78 (m, 4 H), 1.51-1.14 (m, 7 H), 1.08-0.95 (s, 3 H); ESI MS m / z 496 [M + H] ⁺ ; HPLC97.6% (AUC), T R 7.37 _{min; UV (MeOH) λ max 288nm} , ε27,343.
Example 63
4- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -N- (trans-4-((6-hydroxyhexyl) carbamoyl) cyclohexyl) -4H-thieno [3,2-b] Pyrrole-5-carboxamide (JRW-0429)
Step 1. Trans-4- (4- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid (JRW -0427)

According to the general procedure A, trans-4- (4- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane Methyl -1-carboxylate (980 mg, 1.98 mmol) was reacted with lithium hydroxide (142 mg, 5.93 mmol) to give the crude product (1.0 g) as a pale yellow solid. ESI MS m / z 482 [M + H] ^< +>.
Step 2. 4- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -N- (trans-4-((6-hydroxyhexyl) carbamoyl) cyclohexyl) -4H-thieno [3,2 -B] pyrrole-5-carboxamide (JRW-0429)

According to the general procedure B, trans-4- (4- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane -1-Carboxylic acid (200 mg, 0.42 mmol) is reacted with 6-aminohexan-1-ol (73 mg, 0.62 mmol) to give the desired product (200 mg, 83%) as a pale yellow foam The ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.85 (d, J = 8.0, ¹ H), 7.63 (t, J = 5.6, ¹ H), 7.46-7.18 ( m, 5H), 7.11-7.04 (m, 2H), 4.96 (s, 2H), 4.27 (t, J = 5.2, 1H), 3.72-3.55 ( m, 3H), 3.40-3.22 (m, 2H), 3.05-2.95 (m, 2H), 2.72-2.62 (m, 2H), 2.10-1. 97 (m, 1 H), 1.89-1.68 (m, 4 H), 1.53-1.14 (m, 15 H), 1.01 (t, J = 6.9, 3 H); ESI MS ^{m / z 581 [M + H} ] +; HPLC> 99% (AUC), T R 5.85 _{min; UV (MeOH) λ max 288nm} , ε25,165.
Example 64
6- (trans-4- (4- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1-carboxamide ) Hexane-1-sulfonic acid sodium (JRW-0432)
Step 1. 4- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -N- (trans-4-((6-iodohexyl) carbamoyl) cyclohexyl) -4H-thieno [3,2 -B] pyrrole-5-carboxamide (JRW-0431)

A solution of imidazole (60 mg, 0.88 mmol), triphenylphosphine (230 mg, 0.88 mmol), and iodine (223 mg, 0.88 mmol) in THF (10 mL) was stirred at room temperature for 10 minutes. 4- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -N- (trans-4-((6-hydroxyhexyl) carbamoyl) cyclohexyl) -4H dissolved in THF (5 mL) -Thieno [3,2-b] pyrrole-5-carboxamide (170 mg, 0.29 mmol) was added. The solution was stirred at room temperature for 1 hour. The reaction was diluted with ethyl acetate and quenched with 10% Na ₂ S ₂ O ₃ solution. The mixture was diluted with ethyl acetate and water and the layers separated. The organic layer was washed with 10% Na ₂ S ₂ O ₃ solution and brine, dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue was partially purified by column chromatography (silica, dichloromethane / methanol) to give the crude product as a white solid. ESI MS m / z 691 [M + H] ^< +>.
Step 2. 2- (trans-4- (4- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane 1-carboxamido) hexane-1-sulfonic acid sodium (JRW-0432)

4- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -N- (trans-4-((6-iodohexyl) carbamoyl) cyclohexyl) -4H-thieno [in ethanol (10 mL) To a solution of 3,2-b] pyrrole-5-carboxamide (0.29 mmol) was added sodium sulfite (184 mg, 1.46 mmol) and water (10 mL). The mixture was heated to 75 ° C. for 3.5 hours. The reaction was concentrated and the residue was purified by column chromatography (silica, dichloromethane / methanol) to give the desired product (205 mg, quant) as a white solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.88 (d, J = 8.3, ¹ H), 7.68 (t, J = 5.4, ¹ H), 7.48-7. 19 ( m, 5H), 7.12-7.04 (m, 2H), 4.95 (s, 2H), 3.71-3.55 (m, 3H), 3.04-2.94 (m, 5H) 2H), 2.74-2.61 (m, 2H), 2.40-2.32 (m, 2H), 2.11-1.97 (m, 1H), 1.88-1.68 (m. m, 4 H), 1.62-1. 14 (m, 15 H), 1.00 (t, J = 6.7, 3 H); ESI MS m / z 645 [M + H] ⁺ ; HPLC> 99% (AUC ), T _R 5.07 min; UV (MeOH) λ _max 288 nm, ε 18, 276.
Example 65
Trans 4- (4- (2- (ethyl (3-methoxyphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) methyl cyclohexane-1-carboxylate JRW-0327)
Step 1. Methyl 4- (2- (ethyl (3-methoxyphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate (JRW-0299)

To a solution of 2- (5- (methoxycarbonyl) -4H-thieno [3,2-b] pyrrol-4-yl) acetic acid (100 mg, 0.42 mmol) in DMF (3 mL) N-ethyl-3- Methoxyaniline (94 mg, 0.63 mmol), HATU (318 mg, 0.84 mmol), and diisopropylethylamine (162 mg, 1.25 mmol) were added. The reaction was heated to 85 ° C. for 18 hours. The reaction mixture was cooled, diluted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue is purified by column chromatography (silica, heptane / ethyl acetate) to give the desired product (97 mg, 62%) as an orange foam. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.37 (t, J = 8.0, ¹ H), 7.29 (d, J = 5.4, 1 H), 7.15 (s, 1 H), 6 .99-6.84 (m, 3 H), 6.81 (d, J = 5.4, 1 H), 5.02 (s, 2 H), 3.86 (s, 3 H), 3.83-3. .69 (m, 5 H), 1.14 (t, J = 7.2, 3 H); ESI MS m / z 373 [M + H] ^< +>.
Step 2. 4- (2- (ethyl (3-methoxyphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (JRW-0324)

Following the general procedure A, methyl 4- (2- (ethyl (3-methoxyphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate (95 mg, 0. 1). 26 mmol) was reacted with lithium hydroxide (18 mg, 0.77 mmol) to give the crude product (88 mg) as a pale yellow solid. ESI MS m / z 359 [M + H] ^< +>.
Step 3. Trans 4- (4- (2- (ethyl (3-methoxyphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) methyl cyclohexane-1-carboxylate JRW-0327)

4- (2- (ethyl (3-methoxyphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (88 mg, 0.25 mmol) according to General Procedure B ) Was reacted with methyl trans-4-aminocyclohexane-1-carboxylate (48 mg, 0.34 mmol) to give the desired product (78 mg, 63%) as a white solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.90 (d, J = 8.3, ¹ H), 7.50-7.31 (m, 2 H), 7.16-6.91 (m, 5H), 5.00 (s, 2H), 3.81 (s, 3H), 3.75 to 3.53 (m, 6H), 2.33 to 2.18 (m, 1H), 2.01 -1.77 (m, 4H), 1.50-1.22 (m, 4H), 1.08-0.95 (m, 3H); ESI MS m / z 498 [M + H] ⁺ ; HPLC 96.4 % (AUC), T _R 6.69 _{min; UV (MeOH) λ max 289nm} , ε28,671.
Example 66
Trans 4- (4- (2- (ethyl (o-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylate methyl (JRW -0330)
Step 1. 4- (2- (ethyl (o-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate methyl (JRW-0300)

To a solution of 2- (5- (methoxycarbonyl) -4H-thieno [3,2-b] pyrrol-4-yl) acetic acid (100 mg, 0.42 mmol) in DMF (3 mL) N-ethyl-2- Methyl aniline (84 mg, 0.63 mmol), HATU (318 mg, 0.84 mmol), and diisopropylethylamine (162 mg, 1.25 mmol) were added. The reaction was heated to 85 ° C. for 18 hours. The reaction mixture was cooled, diluted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue is purified by column chromatography (silica, heptane / ethyl acetate) to give the desired product (72 mg, 48%) as a white foam. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.43-7.21 (m, 5 H), 7.16 (s, 1 H), 6.78 (d, J = 5.4, 1 H), 5.02 (D, J = 16.9, 1H), 4.74 (d, J = 16.9, 1H), 4.15 (dq, J = 7.1, 14.2, 1H), 3.81 (d s, 3 H), 3.24 (dq, J = 7.1, 14.2, 1 H), 1.14 (t, J = 7.1, 4 H); ESI MS m / z 357 [M + H] ^< +>.
Step 2. 4- (2- (ethyl (o-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (JRW-0328)

According to the general procedure A, methyl 4- (2- (ethyl (o-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate (70 mg, 0.20 mmol ) Was reacted with lithium hydroxide (23 mg, 0.98 mmol) to give the crude product (69 mg) as a pale yellow solid. ESI MS m / z 343 [M + H] ^< +>.
Step 3. Trans 4- (4- (2- (ethyl (o-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylate methyl (JRW -0330)

4- (2- (ethyl (o-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (69 mg, 0.25 mmol) according to General Procedure B Was reacted with methyl trans-4-aminocyclohexane-1-carboxylate (46 mg, 0.24 mmol) to give the desired product (77 mg, 79%) as a white foam. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.91 (d, J = 7.9, ¹ H), 7.49-7.29 (m, 5 H), 7.1 1-6.98 (m, 5) 2H), 4.91 (d, J = 16.7, 1H), 4.79 (d, J = 16.7, 1H), 4.06-3.90 (m, 1H), 3.72- 3.53 (m, 4H), 3.14-2.99 (m, 1 H), 2.34 (s, 3 H), 2.31-2.18 (m, 1 H), 2.0-1. 78 (m, 4 H), 1.51-1. 14 (m, 4 H), 0.99 (t, J = 7.1, 3 H); ESI MS m / z 482 [M + H] ⁺ ; HPLC 92.3% (AUC), T _R 6.97 _{min; UV (MeOH) λ max 288nm} , ε29,468.
Example 67
Trans-4- (4- (2- (6-methyl-3,4-dihydroquinolin-1 (2H) -yl) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide ) Methyl cyclohexane-1-carboxylate (JRW-0331)
Step 1. Methyl 4- (2- (6-Methyl-3,4-dihydroquinolin-1 (2H) -yl) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate (JRW-0301)

To a solution of 2- (5- (methoxycarbonyl) -4H-thieno [3,2-b] pyrrol-4-yl) acetic acid (100 mg, 0.42 mmol) in DMF (3 mL) 2,3,4-Tetrahydroquinoline (92 mg, 0.63 mmol), HATU (318 mg, 0.84 mmol), and diisopropylethylamine (162 mg, 1.25 mmol) were added. The reaction was heated to 85 ° C. for 18 hours. The reaction mixture was cooled, diluted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue is purified by column chromatography (silica, heptane / ethyl acetate) to give the desired product (140 mg, 90%) as a white foam. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.31 (d, J = 5.4, ¹ H), 7.18 (s, 1 H), 7.08 to 6.96 (m, 3 H), 6.82 (D, J = 5.4, 1 H), 5.39 (s, 2 H), 3.86 to 3.74 (m, 5 H), 2.82 to 2. 66 (m, 2 H), 2.31 (S, 3 H), 2.05-1.89 (m, 2 H); ESI MS m / z 367 [M + H] ^< +>.
Step 2. 4- (2- (6-Methyl-3,4-dihydroquinolin-1 (2H) -yl) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid JRW-0329)

4- (2- (6-Methyl-3,4-dihydroquinolin-1 (2H) -yl) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5 following the general procedure A -Methyl carboxylate (130 mg, 0.35 mmol) was reacted with lithium hydroxide (42 mg, 1.76 mmol) to give the crude product (120 mg) as an orange solid. ESI MS m / z 355 [M + H] ^< +>.
Step 3. Trans-4- (4- (2- (6-methyl-3,4-dihydroquinolin-1 (2H) -yl) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide ) Methyl cyclohexane-1-carboxylate (JRW-0331)

Following the general procedure B, 4- (2- (6-methyl-3,4-dihydroquinolin-1 (2H) -yl) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5 -Carboxylic acid (140 mg, 0.40 mmol) is reacted with methyl trans-4-aminocyclohexane-1-carboxylate (92 mg, 0.47 mmol) to give the desired product (130 mg, 67%) as a white solid The ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.89 (d, J = 7.7, ¹ H), 7.47 (d, J = 7.7, ¹ H), 7.39 (d, J = 5.3, 1 H), 7. 19 (d, J = 5.3, 1 H), 7.12 (s, 1 H), 7.08-6.88 (m, 2 H), 5. 44 (s, 1 5) 2H), 3.73-3.53 (m, 6H), 2.72 (t, J = 6.6, 2H), 2.33-2.16 (m, 4H), 2.0-1. 74 (m, 6H), 1.50-1.20 (m, 4H); ESI MS m / z 494 [M + H] +; HPLC98.9% (AUC), T R 7.16 min; UV (MeOH) λ _max 287 nm, ε26, 027.
Example 68
Methyl trans-4- (4- (2- (ethyl (p-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylate (JRW -0334)
Step 1. 4- (2- (ethyl (p-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate methyl (JRW-0314)

To a solution of 2- (5- (methoxycarbonyl) -4H-thieno [3,2-b] pyrrol-4-yl) acetic acid (100 mg, 0.42 mmol) in DMF (3 mL) Methyl aniline (68 mg, 0.50 mmol), HATU (318 mg, 0.84 mmol), and diisopropylethylamine (162 mg, 1.25 mmol) were added. The reaction was heated to 85 ° C. for 18 hours. The reaction mixture was cooled, diluted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue is purified by column chromatography (silica, heptane / ethyl acetate) to give the desired product (120 mg, 80%) as a white foam. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.33 to 7.18 (m, 5 H), 7.16 (s, 1 H), 6.80 (d, J = 5.4, 1 H), 4.95 (S, 2H), 3.84 (s, 3H), 3.74 (q, J = 7.2, 3H), 2.40 (s, 3H), 1.12 (t, J = 7.2) , 3H); ESI MS m / z 357 [M + H] ^< +>.
Step 2. 2- (2- (ethyl (p-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (JRW-0332)

Following the general procedure A, methyl 4- (2- (ethyl (p-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate (120 mg, 0.34 mmol ) Was reacted with lithium hydroxide (40 mg, 1.68 mmol) to give the crude product (109 mg) as a white solid. ESI MS m / z 343 [M + H] ^< +>.
Step 3. Methyl trans-4- (4- (2- (ethyl (p-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylate (JRW -0334)

4- (2- (ethyl (p-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (109 mg, 0.32 mmol) according to General Procedure B Was reacted with methyl trans-4-aminocyclohexane-1-carboxylate (74 mg, 0.38 mmol) to give the desired product (131 mg, 85%) as a white foam. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.88 (d, J = 7.9, ¹ H), 7.41 to 7.25 (m, 5 H), 7.12 to 7.03 (m, 5) 2H), 4.95 (s, 2H), 3.72-3.48 (s, 6H), 2.35 (s, 3H), 2.31-2.19 (s, 1H), 2.01 -1.77 (m, 4H), 1.52-1.19 (m, 4H), 1.05-0.94 (m, 3H); ESI MS m / z 482 [M + H] ⁺ ; HPLC 98.0 % (AUC), T _R 7.04 _{min; UV (MeOH) λ max 289nm} , ε27,490.
Example 69
Trans-4- (4- (2- (ethyl (4- (hydroxymethyl) phenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carvone Methyl acid (JRW-0335)
Step 1. 4- (2-((4-(((tert-Butyldimethylsilyl) oxy) methyl) phenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole- Methyl 5-carboxylate (JRW-0320)

To a solution of 2- (5- (methoxycarbonyl) -4H-thieno [3,2-b] pyrrol-4-yl) acetic acid (150 mg, 0.63 mmol) in DMF (3 mL) -Butyldimethylsilyl) oxy) methyl) -N-ethylaniline (166 mg, 0.63 mmol), HATU (477 mg, 1.25 mmol), and diisopropylethylamine (243 mg, 1.88 mmol) were added. The reaction was heated to 85 ° C. for 30 minutes. The reaction mixture was cooled, diluted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue is purified by column chromatography (silica, heptane / ethyl acetate) to give the desired product (165 mg, 54%) as a pale yellow foam. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.31 (d, J = 8.1, 2 H), 7.23-7.11 (m, 3 H), 7.03 (s, 1 H), 6.66 (D, J = 5.3, 1 H), 4.82 (s, 2 H), 4.66 (s, 2 H), 3.73-3.55 (m, 5 H), 0.99 (t, J = 7.2, 3 H), 0.83 (s, 9 H), 0.00 (s, 6 H); ESI MS m / z 487 [M + H] ^< +>.
Step 2. 2- (2- (ethyl (4- (hydroxymethyl) phenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (JRW-0333)

Following the general procedure A, 4- (2-((4-(((tert-butyldimethylsilyl) oxy) methyl) phenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2- b) Methyl pyrrole-5-carboxylate (165 mg, 0.34 mmol) was reacted with lithium hydroxide (40 mg, 1.68 mmol) to give the crude product (110 mg) as a white solid. ESI MS m / z 359 [M + H] ^< +>.
Step 3. Trans-4- (4- (2- (ethyl (4- (hydroxymethyl) phenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carvone Methyl acid (JRW-0335)

According to the general procedure B, 4- (2- (ethyl (4- (hydroxymethyl) phenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (110 mg, 0.31 mmol) was reacted with methyl trans-4-aminocyclohexane-1-carboxylate (71 mg, 0.37 mmol) to give the desired product (169 mg, 91%) as a white solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.89 (d, J = 7.8, ¹ H), 7.52-7.31 (m, 5 H), 7.12-7.05 (m, 5) 2H), 5.30-5.21 (m, 1H), 4.95 (s, 2H), 4.54 (d, J = 5.6, 2H), 3.74-3.51 (m, 5) 6H), 2.32-2.18 (m, 1 H), 2.00-1. 78 (m, 4 H), 1.52-1.21 (m, 4 H), 1.05-0.94 (m. m, 3H); ESI MS m / z 498 [M + H] +; HPLC98.7% (AUC), T R 5.13 _{min; UV (MeOH) λ max 288nm} , ε24,103.
Example 70
Trans 4- (4- (2- (ethyl (3-isopropylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) methyl cyclohexane-1-carboxylate JRW-0460)
Step 1. Methyl 4- (2- (ethyl (3-isopropylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate (JRW-0454)

To a solution of 2- (5- (methoxycarbonyl) -4H-thieno [3,2-b] pyrrol-4-yl) acetic acid (110 mg, 0.46 mmol) in DMF (5 mL) N-ethyl-3- Isopropylaniline (112 mg, 0.69 mmol), HATU (350 mg, 0.92 mmol), and diisopropylethylamine (178 mg, 1.38 mmol) were added. The reaction was heated to 85 ° C. for 1.5 hours. The reaction mixture was cooled, diluted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue is purified by column chromatography (silica, heptane / ethyl acetate) to give the desired product (147 mg, 83%) as a pale orange oil. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.45-7.34 (m, 1 H), 7.31-7.23 (m, 2 H), 7.20-7.11 (m, 3 H), 6 .80 (d, J = 5.4, 1 H), 4.96 (s, 2 H), 3.83-3.71 (m, 5 H), 3.05-2.87 (m, 1 H), 1 .35-1.25 (m, 6H), 1.18-1.09 (m, 3H); ESI MS m / z 385 [M + H] ^< +>.
Step 2. 2- (2- (ethyl (3-isopropylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (JRW-0457)

Following the general procedure A, methyl 4- (2- (ethyl (3-isopropylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate (145 mg, 0. 1). 38 mmol) was reacted with lithium hydroxide (45 mg, 1.89 mmol) to give the crude product (134 mg) as a pale brown solid. ESI MS m / z 371 [M + H] ^< +>.
Step 3. Trans 4- (4- (2- (ethyl (3-isopropylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) methyl cyclohexane-1-carboxylate JRW-0460)

4- (2- (ethyl (3-isopropylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (130 mg, 0.35 mmol) according to General Procedure B ) Was reacted with methyl trans-4-aminocyclohexane-1-carboxylate (102 mg, 0.53 mmol) to give the desired product (135 mg, 75%) as a white solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.91 (d, J = 7.9, ¹ H), 7.48-7.33 (m, 3 H), 7.32-7.19 (m, 7) 2H), 7.12-7.03 (m, 2H), 4.93 (s, 2H), 3.73-3.52 (m, 6H), 3.04-2.87 (m, 1H) , 2.33-2.18 (m, 1 H), 2.00-1.78 (m, 4 H), 1.51-1.18 (m, 10 H), 1.08-0.95 (m, 1) ESI MS m / z 510 [M + H] ⁺ ; HPLC 98.8% (AUC), T _R 7.61 min; UV (MeOH) λ _max 289 nm, ε 23, 93.
Example 71
Trans-4- (4- (2- (ethyl (3- (hydroxymethyl) phenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid Methyl acid (JRW-0461)
Step 1. 4- (2-((3-(((tert-Butyldimethylsilyl) oxy) methyl) phenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole- Methyl 5-carboxylate (JRW-0455)

To a solution of 2- (5- (methoxycarbonyl) -4H-thieno [3,2-b] pyrrol-4-yl) acetic acid (120 mg, 0.50 mmol) in DMF (5 mL) -Butyldimethylsilyl) oxy) methyl) -N-ethylaniline (200 mg, 0.75 mmol), HATU (381 mg, 1.0 mmol), and diisopropylethylamine (194 mg, 1.50 mmol) were added. The reaction was heated to 85 ° C. for 1.5 hours. The reaction mixture was cooled, diluted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue is purified by column chromatography (silica, heptane / ethyl acetate) to give the desired product (156 mg, 63%) as a colorless oil. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.31 (t, J = 7.8, ¹ H), 7.24-7.17 (m, 1 H), 7.18-7.06 (m, 3 H) , 7.02 (s, 1 H), 6.66 (d, J = 5.2, 1 H), 4.82 (s, 2 H), 4.66 (s, 2 H), 3.71-3.54 (M, 5 H), 0.99 (t, J = 6.9, 3 H), 0.83 (s, 9 H), 0.00 (s, 6 H); ESI MS m / z 487 [M + H] ^< +>.
Step 2. 2- (2- (ethyl (3- (hydroxymethyl) phenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (JRW-0458)

Following the general procedure A, 4- (2-((3-(((tert-butyldimethylsilyl) oxy) methyl) phenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2- b) Methyl pyrrole-5-carboxylate (156 mg, 0.32 mmol) was reacted with lithium hydroxide (38 mg, 1.60 mmol) to give the crude product (150 mg) as a white solid. ESI MS m / z 359 [M + H] ^< +>.
Step 3. Trans-4- (4- (2- (ethyl (3- (hydroxymethyl) phenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid Methyl acid (JRW-0461)

Following the General Procedure B, 4- (2- (ethyl (3- (hydroxymethyl) phenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (150 mg, 0.42 mmol) was reacted with methyl trans-4-aminocyclohexane-1-carboxylate (121 mg, 0.63 mmol) to give the desired product (126 mg, 60%) as a white foam. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.90 (d, J = 8.0, ¹ H), 7.54-7.27 (m, 5 H), 7.13-7.03 (m, 5) 2H), 5.33-5.24 (s, 1 H), 4.96 (s, 2 H), 4.56 (d, J = 5.5, 2 H), 3.73-3.52 (m, 5) 6H), 2.31-2.18 (m, 1 H), 1.99-1.78 (m, 4 H), 1.53-1.21 (m, 4 H), 1.07-0.94 (m) m, 3H); ESI MS m / z 498 [M + H] +; HPLC97.2% (AUC), T R 5.23 _{min; UV (MeOH) λ max 289nm} , ε25,856.
Example 72
Trans-4- (4- (2-((3- (bromomethyl) phenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1- Methyl carboxylate (JRW-0466)

To a solution of carbon tetrabromide (333 mg, 1.0 mmol) and triphenylphosphine (263 mg, 1.0 mmol) in THF (5 mL), trans-4- (4- (2- (ethyl) in THF (5 mL) (3- (Hydroxymethyl) phenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) methyl cyclohexane-1-carboxylate (50 mg, 0.10 mmol) was added . The reaction was stirred at room temperature for 48 hours. The mixture was diluted with DCM and washed with water. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue was purified by column chromatography (silica, dichloromethane / methanol) to give the desired product (11 mg, 20%) as a white solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.94 to 7.86 (m, 1 H), 7.72 to 7.30 (m, 5 H), 7.19 to 6.97 (m, 2 H) , 4.96 (s, 2H), 4.74 (s, 2H), 3.76-3.49 (m, 6H), 2.37-2.17 (m, 1H), 2.05-1 .75 (m, 4H), 1.55 to 1.19 (m, 4H), 1.10 to 0.95 (m, 3H); ESI MS m / z 562 [M + H] ⁺ ; HPLC 87.2% ( AUC), T _R 6.96 _{min; UV (MeOH) λ max 289nm} , ε20,648.
Example 73
Trans-4- (4- (2-((3- (dimethylamino) phenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1 -Methyl carboxylate (JRW-0478)
Step 1. Methyl 4- (2-((3- (dimethylamino) phenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate (JRW-0475 )

To a solution of 2- (5- (methoxycarbonyl) -4H-thieno [3,2-b] pyrrol-4-yl) acetic acid (115 mg, 0.48 mmol) in DMF (3 mL) was added N1-ethyl-N3,. N3-Dimethylbenzene-1,3-diamine (118 mg, 0.72 mmol), HATU (365 mg, 0.96 mmol), and diisopropylethylamine (186 mg, 1.44 mmol) were added. The reaction was heated to 85 ° C. for 1 hour. The reaction mixture was cooled, diluted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue is purified by column chromatography (silica, heptane / ethyl acetate) to give the desired product (152 mg, 82%) as a yellow solid. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.35 to 7.23 (m, 2 H), 7.14 (s, 1 H), 6.85 to 6.60 (m, 4 H), 5.05 (s) , 3H), 3.89-3.61 (m, 5H), 3.02 (s, 6H), 1.29-0.97 (m, 3H); ESI MS m / z 386 [M + H] ^< +>.
Step 2. 4- (2-((3- (Dimethylamino) phenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (JRW-0476)

Following general procedure A, methyl 4- (2-((3- (dimethylamino) phenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate Reaction (160 mg, 0.42 mmol) with lithium hydroxide (50 mg, 2.1 mmol) gave the crude product (130 mg) as a pale green solid. ESI MS m / z 372 [M + H] ^< +>.
Step 3. Trans-4- (4- (2-((3- (dimethylamino) phenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1 -Methyl carboxylate (JRW-0478)

Following the general procedure B, 4- (2-((3- (dimethylamino) phenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid ( 130 mg (0.35 mmol) was reacted with methyl trans-4-aminocyclohexane-1-carboxylate (102 mg, 0.53 mmol) to give the desired product (132 mg, 74%) as an off white solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.91 (d, J = 7.9, ¹ H), 7.35 (d, J = 5.4, ¹ H), 7.28 (t, J = 7.9, 1 H), 7.11-7.04 (m, 2 H), 6.86-6. 65 (m, 3 H), 5.01 (s, 2 H), 3.74-3.51 (m. m, 6H), 2.95 (s, 6H), 2.32-2.19 (s, 1H), 2.01-1.79 (m, 4H), 1.49-1.25 (m, 5) 4H), 1.00 (t, J = 7.1,3H); ESI MS m / z 511 [M + H] +; HPLC97.8% (AUC), T R 5.21 min; UV (MeOH) λ _max 289 nm, ε29, 909.
Example 74
Methyl trans-4- (4- (2- (ethyl (3-isobutylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylate JRW-0508)
Step 1. 4- (2- (Ethyl (3-isobutylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate methyl (JRW-0502)

To a solution of 2- (5- (methoxycarbonyl) -4H-thieno [3,2-b] pyrrol-4-yl) acetic acid (100 mg, 0.42 mmol) in DMF (3 mL) N-ethyl-3- Isobutylaniline (89 mg, 0.50 mmol), HATU (318 mg, 0.84 mmol), and diisopropylethylamine (162 mg, 1.25 mmol) were added. The reaction was heated to 85 ° C. for 4 hours. The reaction mixture was cooled, diluted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue is purified by column chromatography (silica, heptane / ethyl acetate) to give the desired product (128 mg, 77%) as a thick oil. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.38 (t, J = 7.7, ¹ H), 7.28 (d, J = 5.4, ¹ H), 7.21-7.13 (m, 3H), 7.13-7.09 (m, 1H), 6.79 (d, J = 4.8, 1H), 4.95 (s, 2H), 3.84-3.69 (m, 5) 5H), 2.54 (d, J = 7.2, 2H), 1.98-1.82 (m, 1H), 1.13 (t, J = 7.2, 3H), 0.92 ( t, J = 6.1, 6H); ESI MS m / z 399 [M + H] ^< +>.
Step 2. 2- (2- (ethyl (3-isobutylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (JRW-0505)

Following the general procedure A, methyl 4- (2- (ethyl (3-isobutylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate (120 mg, 0. 1). 30 mmol) was reacted with lithium hydroxide (36 mg, 1.5 mmol) to give the crude product (125 mg) as a pale yellow solid. ESI MS m / z 385 [M + H] ^< +>.
Step 3. Methyl trans-4- (4- (2- (ethyl (3-isobutylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylate JRW-0508)

4- (2- (ethyl (3-isobutylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylic acid (120 mg, 0.42 mmol) according to General Procedure B ) Was reacted with methyl trans-4-aminocyclohexane-1-carboxylate (90 mg, 0.47 mmol) to give the desired product (130 mg, 80%) as a white solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.90 (d, J = 7.9, ¹ H), 7.47-7.13 (m, 5 H), 7.12-7.04 (m, 5) 2H), 4.93 (s, 2H), 3.72 to 3.52 (m, 6H), 2.57 to 2.46 (m, 2H), 2.32 to 2.19 (m, 1H) , 2.00-1.79 (m, 5H), 1.50-1.20 (m, 4H), 1.06-0.94 (m, 3H), 0.86 (d, J = 6. 6,6H); ESI MS m / z 524 [M + H] +; HPLC> 99% (AUC), T R 6.73 _{min; UV (MeOH) λ max 289nm} , ε19,115.
Scheme 6.
Example 75
Trans-4- (1- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -1H-indole-2-carboxamido) cyclohexane-1-carboxylate methyl (JRW-0355)
Step 1. Methyl 1- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -1H-indole-2-carboxylate (JRW-0349)

Potassium carbonate (94 mg, 0.68 mmol), 18-crown-6 ether (7 mg, 0.03 mmol), in a solution of methyl 1H-indole-2-carboxylate (100 mg, 0.57 mmol) in acetonitrile (5 mL), And 2-chloro-N-ethyl-N- (m-tolyl) acetamide (120 mg, 0.57 mmol) were added. The suspension was heated to 75 ° C. for 18 hours. The mixture was diluted with ethyl acetate and water and the layers separated. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue is purified by column chromatography (silica, heptane / ethyl acetate) to give the desired product (108 mg, 54%) as a white solid. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.65 (d, J = 8.0, ¹ H), 7.44-7.07 (m, 8 H), 5.05 (s, 2 H), 3.88 (S, 3 H), 3.78-3. 68 (m, 2 H), 2.43 (s, 3 H), 1. 12 (t, J = 7.2, 3 H); ESI MS m / z 351 [ M + H] ⁺ .
Step 2. 2- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -1H-indole-2-carboxylic acid (JRW-0353)

According to General Procedure A, methyl 1- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -1H-indole-2-carboxylate (108 mg, 0.31 mmol) in lithium hydroxide (37 mg, Reaction with 1.54 mmol) gave the crude product (103 mg) as a white solid. ESI MS m / z 337 [M + H] ^< +>.
Step 3. Trans-4- (1- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -1H-indole-2-carboxamido) cyclohexane-1-carboxylate methyl (JRW-0355)

According to the general procedure B, 1- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -1H-indole-2-carboxylic acid (103 mg, 0.31 mmol) with trans-4-aminocyclohexane Reaction with methyl 1-carboxylate (71 mg, 0.37 mmol) gave the desired product (133 mg, 91%) as a white foam. ESI MS m / z 476 [M + H] ^< +>.
Example 76
Trans-4- (1- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -1H-indole-2-carboxamide) methyl cyclohexane-1-carboxylate (JRW-0424)
Step 1. 2-Chloro-N-ethyl-N- (3-ethylphenyl) acetamide (JRW-0413)

To a solution of N-ethyl-3-ethylaniline (0.97 g, 6.50 mmol) in ethyl acetate (30 mL) was added water (10 mL). The biphasic solution was cooled to 0 ° C. and potassium hydroxide (1.09 g, 19.5 mmol) was added in one portion. 2-Chloroacetyl chloride (1.10 g, 0.76 mL, 9.75 mmol) was added dropwise over 10 minutes. The mixture was stirred for 1 hour, diluted with water and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated to give the crude product (1.54 g) as mobile oil. ESI MS m / z 226 [M + H] ^< +>.
Step 2. Methyl 2- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -1H-indole-2-carboxylate (JRW-0416)

Potassium carbonate (551 mg, 4.0 mmol), 18-crown-6 ether (44 mg, 0.17 mmol) in a solution of methyl 1H-indole-2-carboxylate (582 mg, 3.3 mmol) in acetonitrile (20 mL), 2-Chloro-N-ethyl-N- (3-ethylphenyl) acetamide (750 mg, 3.3 mmol) was added. The suspension was heated to 75 ° C. for 18 hours. The mixture was diluted with ethyl acetate and water and the layers separated. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue was purified by column chromatography (silica, heptane / ethyl acetate) to give the desired product (0.74 g, 61%) as a pale brown solid. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.65 (d, J = 8.1, ¹ H), 7.50-7.07 (m, 8 H), 5.06 (s, 2 H), 3.87 (S, 3H), 3.82-3.69 (m, 4H), 2.78-2.63 (m, 2H), 1.35-1.24 (m, 3H), 1.18-1 .06 (m, 3 H); ESI MS m / z 365 [M + H] ^< +>.
Step 3. 3- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -1H-indole-2-carboxylic acid (JRW-0419)

Following general procedure A, methyl 1- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -1H-indole-2-carboxylate (740 mg, 2.0 mmol) in lithium hydroxide (243 mg) The reaction with 10.1 mmol) gave the crude product (690 mg) as a white solid. ESI MS m / z 350 [M + H] ^< +>.
Step 4. Trans-4- (1- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -1H-indole-2-carboxamide) methyl cyclohexane-1-carboxylate (JRW-0424)

Following the General Procedure B, 1- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -1H-indole-2-carboxylic acid (690 mg, 1.97 mmol) to trans-4-aminocyclohexane Reaction with methyl 1-carboxylate (457 mg, 2.36 mmol) gave the desired product (760 mg, 79%) as a white foam. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 8.20 (d, J = 7.9, ¹ H), 7.58 (d, J = 7.9, ¹ H), 7.51-7. m, 6H), 7.13-7.02 (m, 2H), 5.02 (s, 2H), 3.77-3.54 (m, 6H), 2.69 (d, J = 7.). 6, 2 H), 2.33-2.21 (m, 1 H), 2.02-1.81 (m, 4 H), 1.52-1.30 (m, 4 H), 1.23 (t, 2) J = 7.6,3H), 1.08-0.95 (m , 3H); ESI MS m / z 490 [M + H] +; HPLC> 99% (AUC), T R 7.56 min; UV ( MeOH) λ _max 291 nm, ε 15, 737.
Example 77
1- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -N- (trans-4-((6-hydroxyhexyl) carbamoyl) cyclohexyl) -1H-indole-2-carboxamide (JRW- 0430)
Step 1. Trans-4- (1- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -1H-indole-2-carboxamido) cyclohexane-1-carboxylic acid (JRW-0428)

According to the general procedure A, methyl trans-4- (1- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -1H-indole-2-carboxamido) cyclohexane-1-carboxylate (720 mg , 1.47 mmol) was reacted with lithium hydroxide (105 mg, 4.4 mmol) to give the crude product (680 mg) as a white solid. ESI MS m / z 476 [M + H] ^< +>.
Step 2. 2- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -N- (trans-4-((6-hydroxyhexyl) carbamoyl) cyclohexyl) -1H-indole-2-carboxamide (JRW-0430)

According to the general procedure B, trans-4- (1- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -1H-indole-2-carboxamido) cyclohexane-1-carboxylic acid (200 mg, 0.42 mmol) was reacted with 6-aminohexan-1-ol (74 mg, 0.63 mmol) to give the desired product (218 mg, 79%) as a pale yellow foam. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 8.17 (d, J = 8.0, ¹ H), 7.69-7.54 (m, 2 H), 7. 50-7.14 (m, 6H), 7.13 to 7.02 (m, 2H), 5.02 (s, 2H), 4.27 (t, J = 5.2, 1H), 3.77 to 3.52 (s, 7 2) 3H), 3.36 (dd, J = 6.4, 11.7, 2H), 3.05-2.95 (m, 2H), 2.74-2.63 (m, 2H), 2.H. 13-1.97 (m, 1H), 1.91-1.69 (m, 4H), 1.54-1.15 (m, 15H), 1.09-0.93 (m, 3H); ESI MS m / z 575 [M + H] +; HPLC> 99% (AUC), T R 6.12 _{min; UV (MeOH) λ max 291nm} , ε17,243.
Example 78
6- (trans-4- (1- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -1H-indole-2-carboxamido) cyclohexane-1-carboxamido) hexane-1-sulfonic acid sodium (JRW-0434)
Step 1. 1- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -N- (trans-4-((6-iodohexyl) carbamoyl) cyclohexyl) -1H-indole-2-carboxamide (JRW-0433)

A solution of imidazole (66 mg, 0.97 mmol), triphenylphosphine (254 mg, 0.97 mmol), and iodine (246 mg, 0.97 mmol) in THF (10 mL) was stirred at room temperature for 10 minutes. 1- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -N- (trans-4-((6-hydroxyhexyl) carbamoyl) cyclohexyl) -1H dissolved in THF (5 mL) -Indole-2-carboxamide (186 mg, 0.32 mmol) was added. The solution was stirred at room temperature for 1 hour. The reaction was diluted with ethyl acetate and quenched with 10% Na ₂ S ₂ O ₃ solution. The mixture was diluted with ethyl acetate and water and the layers separated. The organic layer was washed with 10% Na ₂ S ₂ O ₃ solution and brine, dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue was partially purified by column chromatography (silica, dichloromethane / methanol) to give the crude product as a white solid. ESI MS m / z 685 [M + H] ^< +>.
Step 2. 2- (trans-4- (1- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -1H-indole-2-carboxamido) cyclohexane-1-carboxamido) hexane-1- Sodium sulfonate (JRW-0434)

1- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -N- (trans-4-((6-iodohexyl) carbamoyl) cyclohexyl) -1H-indole- in ethanol (10 mL) To a solution of 2-carboxamide (0.32 mmol) was added sodium sulfite (203 mg, 1.6 mmol) and water (10 mL). The mixture was heated to 75 ° C. for 2 hours. The reaction is concentrated and the residue is purified by column chromatography (silica, dichloromethane / methanol) to give the desired product (190 mg, 89%) as a white solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 8.21 (d, J = 8.2, 1 H), 7.69 (t, J = 5.6, ¹ H), 7.58 (d, J = 7.8, 1 H), 7.5 1 to 7. 14 (m, 6 H), 7.1 2 to 7.0 3 (m, 2 H), 5.0 2 (s, 2 H), 3.76 to 3.53 (m) m, 3H), 3.00 (dd, J = 6.6, 12.7, 2H), 2.76-2.61 (m, 2H), 2.39-2.32 (m, 2H), 2.12 to 1.99 (m, 1 H), 1.90 to 1. 70 (m, 4 H), 1.61 to 1. 14 (m, 15 H), 1.08 to 0.95 (s, 3 H) ); ESI MS m / z 639 [M + H] +; HPLC> 99% (AUC), T R 5.07 _{min; UV (MeOH) λ max 291nm} , ε15,800.
Example 79
Trans-4- (1- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -6-methoxy-1H-indole-2-carboxamido) cyclohexane-1-carboxylate methyl (JRW-0359)
Step 1. Methyl 1- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -6-methoxy-1H-indole-2-carboxylate (JRW-0351)

To a solution of methyl 6-methoxy-1H-indole-2-carboxylate (116 mg, 0.57 mmol) in acetonitrile (5 mL), potassium carbonate (94 mg, 0.68 mmol), 18-crown-6 ether (7 mg, 0 .03 mmol), and 2-chloro-N-ethyl-N- (m-tolyl) acetamide (120 mg, 0.57 mmol) were added. The suspension was heated to 70 ° C. for 2 days. The mixture was diluted with ethyl acetate and water and the layers separated. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue is purified by column chromatography (silica, heptane / ethyl acetate) to give the desired product (155 mg, 72%) as a white solid. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.51 (d, J = 8.7, ¹ H), 7.41-7.31 (m, 1 H), 7.24-7.08 (m, 4 H) , 6.79 (dd, J = 2.2, 8.7, 1 H), 6.62-6.55 (m, 1 H), 5.01 (s, 2 H), 3.87 (s, 3 H) , 3.84 (s, 3 H), 3.81-3. 68 (m, 2 H), 2.42 (s, 3 H), 1. 18-1.09 (m, 3 H); ESI MS m / z 381 [M + H] ⁺ .
Step 2. 2- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -6-methoxy-1H-indole-2-carboxylic acid (JRW-0357)

According to General Procedure A, methyl 1- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -6-methoxy-1H-indole-2-carboxylate (150 mg, 0.39 mmol) is hydroxylated Reaction with lithium (47 mg, 1.97 mmol) gave the crude product (150 mg) as a white solid. ESI MS m / z 367 [M + H] ^< +>.
Step 3. Trans-4- (1- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -6-methoxy-1H-indole-2-carboxamido) cyclohexane-1-carboxylate methyl (JRW-0359)

Following General Procedure B, trans 4- (1- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -6-methoxy-1H-indole-2-carboxylic acid (150 mg, 0.41 mmol) Reaction with methyl aminocyclohexane-1-carboxylate (95 mg, 0.49 mmol) gave the desired product (163 mg, 78%) as a white foam. ESI MS m / z 506 [M + H] ^< +>.
Example 80
Methyl trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-furo [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylate (JRW -360)
Step 1. Methyl 4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-furo [3,2-b] pyrrole-5-carboxylate (JRW-0352)

Potassium carbonate (94 mg, 0.68 mmol), 18-crown-6 ether, to a solution of methyl 4H-furo [3,2-b] pyrrole-5-carboxylate (94 mg, 0.57 mmol) in acetonitrile (5 mL) (7 mg, 0.03 mmol) and 2-chloro-N-ethyl-N- (m-tolyl) acetamide (120 mg, 0.57 mmol) were added. The suspension was heated to 70 ° C. for 2 days. The mixture was diluted with ethyl acetate and water and the layers separated. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue is purified by column chromatography (silica, heptane / ethyl acetate) to give the desired product (166 mg, 86%) as a white solid. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.47 (d, J = 2.2, ¹ H), 7.35 (t, J = 7.7, ¹ H), 7.24-7.17 (m, 1H), 7.16 to 7.07 (m, 2H), 6.81 to 6.78 (m, 1H), 6.38 to 6.36 (m, 1H), 4.86 (s, 2H) , 3.85-3.67 (m, 5H), 2.40 (s, 3H), 1.12 (t, J = 7.2, 3H); ESI MS m / z 341 [M + H] ^< +>.
Step 2. 4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-furo [3,2-b] pyrrole-5-carboxylic acid (JRW-358)

Following the general procedure A, methyl 4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-furo [3,2-b] pyrrole-5-carboxylate (160 mg, 0.47 mmol ) Was reacted with lithium hydroxide (56 mg, 2.35 mmol) to give the crude product (150 mg) as a white solid. ESI MS m / z 327 [M + H] ^< +>.
Step 3. Methyl trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-furo [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylate (JRW -360)

4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-furo [3,2-b] pyrrole-5-carboxylic acid (150 mg, 0.46 mmol) according to General Procedure B Was reacted with methyl trans-4-aminocyclohexane-1-carboxylate (106 mg, 0.55 mmol) to give the desired product (178 mg, 83%) as a white foam. ESI MS m / z 466 [M + H] ^< +>.
Example 81
Trans-4- (1- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -1H-pyrrole-2-carboxamide) methyl cyclohexane-1 -carboxylate (JRW-0456)
Step 1. Methyl 1- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -1H-pyrrole-2-carboxylate (JRW-0450)

Potassium carbonate (391 mg, 2.83 mmol), 18-crown-6 ether (31 mg, 0.12 mmol), in a solution of methyl 1H-pyrrole-2-carboxylate (295 mg, 2.36 mmol) in acetonitrile (20 mL) And 2-chloro-N-ethyl-N- (m-tolyl) acetamide (500 mg, 2.36 mmol) were added. The suspension was heated to 75 ° C. for 18 hours. The mixture was diluted with ethyl acetate and water and the layers separated. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue is purified by column chromatography (silica, heptane / ethyl acetate) to give the desired product (530 mg, 75%) as a clear oil. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.35 (t, J = 7.5, ¹ H), 7.24-7.10 (m, 3 H), 6.93 (dd, J = 1.8, 3.9, 1 H), 6.78-6. 70 (m, 1 H), 6. 13 (dd, J = 2.6, 3.9, 1 H), 4.77 (s, 2 H), 3. 85-3.61 (m, 6H), 2.41 (s, 3H), 1.13 (t, J = 7.2, 3H); ESI MS m / z 301 [M + H] ^< +>.
Step 2. 2- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -1H-pyrrole-2-carboxylic acid (JRW-0453)

According to General Procedure A, methyl 1- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -1H-pyrrole-2-carboxylate (530 mg, 1.76 mmol) in lithium hydroxide (211 mg, Reaction with 8.82 mmol) gave the crude product (475 mg) as a white solid. ESI MS m / z 287 [M + H] ^< +>.
Step 3. Trans-4- (1- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -1H-pyrrole-2-carboxamide) methyl cyclohexane-1 -carboxylate (JRW-0456)

According to the general procedure B, 1- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -1H-pyrrole-2-carboxylic acid (475 mg, 1.66 mmol) with trans-4-aminocyclohexane Reaction with methyl 1-carboxylate (482 mg, 2.49 mmol) gave the desired product (505 mg, 71%) as a white foam. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.62 (d, J = 8.0, 1 H), 7.41 to 7.30 (m, 1 H), 7.26 to 7.12 (m, 7) 3H), 6.82-6.67 (m, 2H), 5.94 (dd, J = 2.6, 3.8, 1H), 4.77 (s, 2H), 3.67-3. 54 (m, 6 H), 2.34 (s, 3 H), 2.29-2.18 (s, 1 H), 1.98-1.74 (m, 4 H), 1.48-1.26 ( m, 4H), 1.09-0.92 (m , 3H); ESI MS m / z 426 [M + H] +; HPLC97.5% (AUC), T R 6.05 min; UV (MeOH) λ _max 264 nm, ε11, 978.
Example 82
Trans-4- (6- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -6H-thieno [2,3-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylate methyl (JRW -0463)
Step 1.6- (2- (Ethyl (m-tolyl) amino) -2-oxoethyl) -6H-thieno [2,3-b] pyrrole-5-carboxylate methyl (JRW-0459)

Potassium carbonate (157 mg, 1.1 mmol), 18-crown-6 ether, in a solution of methyl 6H-thieno [2,3-b] pyrrole-5-carboxylate (171 mg, 0.94 mmol) in acetonitrile (10 mL) (13 mg, 0.047 mmol), and 2-chloro-N-ethyl-N- (m-tolyl) acetamide (200 mg, 0.94 mmol) were added. The suspension was heated to 75 ° C. for 18 hours. The mixture was diluted with ethyl acetate and water and the layers separated. The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was then concentrated. The residue is purified by column chromatography (silica, heptane / ethyl acetate) to give the desired product (260 mg, 77%) as a white solid. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.37 (t, J = 7.6, ¹ H), 7.25-7.09 (m, 4 H), 6.99-6. 95 (m, 1 H) , 6.91-6.85 (m, 1 H), 4.93 (s, 2 H), 3.92-3.64 (m, 5 H), 2.43 (s, 3 H), 1.14 (t , J = 7.2, 4 H); ESI MS m / z 357 [M + H] ^< +>.
Step 2. 2- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -6H-thieno [2,3-b] pyrrole-5-carboxylic acid (JRW- 0462)

Following the general procedure A, methyl 6- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -6H-thieno [2,3-b] pyrrole-5-carboxylate (260 mg, 0.73 mmol ) Was reacted with lithium hydroxide (87 mg, 3.6 mmol) to give the crude product (240 mg) as a white solid. ESI MS m / z 342 [M + H] ^< +>.
Step 3. Trans-4- (6- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -6H-thieno [2,3-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylate methyl (JRW -0463)

6- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -6H-thieno [2,3-b] pyrrole-5-carboxylic acid (240 mg, 0.70 mmol) according to General Procedure B Was reacted with methyl trans-4-aminocyclohexane-1-carboxylate (203 mg, 1.05 mmol) to give the desired product (320 mg, 95%) as a pale yellow foam. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.88 (d, J = 8.0, ¹ H), 7.44-7.33 (m, 1 H), 7.28-7.14 (m, 3H), 7.11 to 6.95 (m, 3H), 4.90 (s, 2H), 3.72 to 3.52 (m, 6H), 2.36 (s, 3H), 2.25 (S, 1 H), 2.02-1. 76 (m, 4 H), 1.51-1. 20 (m, 4 H), 1.00 (t, J = 6.9, 3 H); ESI MS m ^{/ z 482 [M + H]} +; HPLC> 99% (AUC), T R 7.01 _{min; UV (MeOH) λ max 287nm} , ε12,894.

Example 83
Inhibition of Nluc Luciferase K562 was plated at 2000 cells / well in wells of a Corning 3707 assay plate in 20 μL RPMI medium and incubated overnight. Trail titrations were prepared in 10 μL RPMI medium and added to cells. Thereafter, 10 μL of either 4 × REALTIME-GLOTM MT Cell Viability Reagent (RTCV; Promega Corporation) or media (control) were added to each well. The reactions were incubated at 37 ° C./5% CO ₂ at various times and luminescence was measured on a Tecan M1000 Pro plate reader. At 5 hours, 40 μL of CASPASE-GLO® 3/7 detection reagent (Promega Corporation) was added with or without 200 μM JRW-0004. Reactions were incubated for 2 hours at room temperature (RT) and luminescence was measured on a Tecan M1000 Pro plate reader. FIG. 1 shows in a multiplex that JRW-0004 combines the REALTIME-GLOTM assay and the CASPASE-GLOTM assay to inhibit the Nluc enzyme in the REALTIME-GLOTM MT cell viability reagent. , CASPASE-GLO® assay window can be restored.

Example 84
Inhibition of Purified Nluc Luciferase In Corning 3570 assay plates, purified Nluc enzyme (Promega Corporation), 20 mM DTT, and Nluc Pro substrate converted to Nluc substrate upon reduction by DTT (US Patent Publication No. 2013/0130289) PBI-4442) described in the above were added. Then, compound titration of JRW-0004, JRW-0013, JRW-0006, JRW-0042, JRW-0138, and JRW-0147 was performed (1: 2 serial dilution in PBS, 11 points + no inhibitor) Control, starting at 200 μM; final concentration of 100 μM in the reaction). Compound titrations were added in equal volumes to the wells of the assay plate. Reactions were incubated for 2 hours at room temperature and luminescence was measured on a Tecan M1000 Pro plate reader. The half maximal inhibition (thienopyrrole compound) concentration (IC ₅₀ ) was determined using GraphPad Prism 6.03. FIG. 2 shows that all compounds can inhibit purified Nluc enzyme.

Example 85
Specificity of Inhibition of Nluc Luciferase The following example illustrates Nluc luciferase activity versus firefly luciferase activity, eg, thienopyrrole compounds JRW-0004, JRW-0013, JRW-0006 of the present disclosure for inhibiting ULTRA GLO® luciferase. , JRW-0042, JRW-0138, and JRW-0147 are described. For Corning 3570 assay plates, a solution containing 1 μM luciferin in luciferase detection reagent (Promega Corporation V865 / 859) was added to the assay wells. Thereafter, titrations of equal volumes of thienopyrrole compounds JRW-0004, JRW-0013, JRW-0006, JRW-0042, JRW-0138, and JRW-0147 were added to the wells. Reactions were incubated for 2 hours at room temperature and luminescence was measured on a Tecan M1000 Pro plate reader. FIG. 3 shows that the thienopyrrole compound did not inhibit firefly luciferase activity.

Example 86
Thienopyrrole Compounds that Enable Multiplexing The following example describes the use of thienopyrrole compounds of the present invention to enable multiplexing of an assay that utilizes Nluc luciferase and another luciferase, such as firefly luciferase.

  A) In Corning 3570 Assay Plate, MCF7 cells are plated at 1,000 cells / well in 20 μL of cell culture medium (EMEM supplemented with 0.01 mg / mL human recombinant insulin and 10% fetal bovine serum) Incubated overnight. 10 μL of 4 × REALTIME-GLOTM MT cell viability reagent (Promega Corporation) or 10 μL medium alone in media was added to the cells. 10 μL of 40 μM staurosporine in culture medium or 10 μL medium alone (control) was added to the cells. The cells were incubated and caspase activation was monitored 5.5 hours after reagent addition. To monitor caspase activation, 40 μL of CASPASE-GLO® 3/7 assay reagent (Promega Corporation) as it is (caspase) or with 200 μM of JRW-0004, JRW-0013, or JRW-0042. Either was added. Reactions were incubated at room temperature and luminescence was measured on a Tecan M1000 Pro plate reader one hour and ten minutes after addition of reagents.

Table 1 shows that the compounds inhibit background luminescence from REALTIME-GLOTM in multiplexing with CASPASE-GLO® (medium containing REALTIME-GLOTM). When comparing the signal generated from the CASPASE-GLO® reagent in medium alone to the signal generated in the medium containing the REALTIME-GLOTM reagent, the signal is higher in the multiplex reaction . The compounds inhibit the Nluc enzyme and reduce background luminescence from the REALTIME-GLOTM reagent.
Table 1

  B) A549 cells were plated in wells of Corning 3570 plates (n-4) at 1,000 cells / well in 20 μL F12K medium and incubated overnight. The REALTIME-GLOTM MT Cell Viability Assay Reagent (Promega Corporation) was then added to the wells as a 2 × solution in 20 μL medium. The reaction was incubated for 1 hour. A 40 μL titration of JRW-0013 or JRW-0147 in CASPASE-GLO® 3/7 assay reagent (Promega Corporation) was added at 2 × concentration. Reactions were incubated at room temperature and luminescence was assessed at 1 hour.

Table 2 shows that these compounds can dose dependently inhibit the Nluc enzyme in the REALTIME-GLOTM MT cell viability assay in multiplexing with the CASPASE-GLO® assay.
Table 2

Example 87
The following examples show that compounds JRW-0004, WZ141-88, WZ141-86, WZ141-74, WZ141-84, WZ141-89, WZ141-90, and WZ141-91 inhibit Nluc luciferase. A solution of purified Nluc and Nluc pro-substrate (PBI-4442 described in US2013 / 0130289), which is converted to Nluc substrate upon reduction by DTT, was prepared to pH 7.5 in PBS buffer. A 40 mM solution of DTT pH 7.5 in PBS containing 1% TERGITOL was also prepared. Then also titration of the thienopyrrole compounds JRW-0004, WZ141-88, WZ141-86, WZ141-74, WZ141-84, WZ141-89, WZ141-90, and WZ141-91 in buffer containing DTT and TERGITOL Prepared. An equal volume of Nluc / pre-substrate solution was added to the thienopyrrole compound titration in the wells of the assay plate. Reactions were incubated at room temperature and luminescence was measured at various time points on a Tecan M100 Pro plate reader (integration time 200 ms). IC ₅₀ values were determined using GraphPad Prism 6.03. Figures 4 to 6 show that all compounds tested inhibit Nluc luciferase in a dose and time dependent manner.

Example 88
Cell Permeability The following example demonstrates the permeability of the thienopyrrole compounds described herein. HEK 293 or HeLa cells were transiently transfected with β2 adrenergic receptor-Nluc (B2AR-Nluc) fusion protein to cytoplasmically fix and orient Nluc. Twenty-four hours after transfection, cells were treated with − / + 50 μg / mL digitonin to mimic the live and lytic scenarios, respectively. The intact and permeabilized cells were then exposed to the compound response curve of the thienopyrrole compound for up to 2 hours. 10 μM flimazine (Promega Corp.) was added and luminescence was measured. When thienopyrrole compounds (inhibitors) were permeable, both live and permeable cell dose response curves overlapped. However, when the thienopyrrole compound was impermeable, the EC ₅₀ shifted to the right in living cells compared to permeable cells. See Figures 7-11.

Example 89
Inhibition of Extracellular BRET The following example demonstrates the ability of the thienopyrrole compounds described herein to inhibit extracellular BRET. See Figure 12A. HEK293 was transiently transfected with Nluc-HDAC6 fusion protein to orient Nluc-HDAC6 inside the cells. Twenty four hours after transfection, cells were treated with 10 ng / mL of purified Nluc-HALOTAG labeled with NANOBRET 618 ligand to simulate mock extracellular BRET. The cells were then exposed to the compound response curves of the thienopyrrole compounds JRW-0013, JRW-0051, JRW-0147, and JRW-0187 for 2 hours. After that, 10 μM flimazine was added, and a BRET ratio of 610/450 nm was measured. When thienopyrrole compounds, such as JRW-0013, were permeable, the BRET ratio remained constant throughout the compound dose response curve. When the thienopyrrole compound, eg, JRW-0051, JRW-0147, or JRW-0187, was impermeable, the BRET ratio decreased across the compound dose response curve and intracellular Nluc signal while inhibiting extracellular BRET. Was enhanced. See Figures 12B-12E.

Example 90
Inhibition of Extracellular Luciferase Activity and Enhancement of Intracellular BRET The following example demonstrates the ability of the thienopyrrole compounds described herein to inhibit extracellular luciferase activity while enhancing intracellular BRET. See Figure 13A. HEK293 was transiently transfected with Nluc-HALOTAG fusion protein to orient Nluc-HALOTAG inside the cells. Twenty-four hours after transfection, cells were labeled with NANOBRET 618 ligand to simulate specific intracellular BRET. Cells were then treated with 10 ng / mL of purified Nluc to simulate simulated extracellular luminescence. Cells were exposed to the compound response curves of the thienopyrrole compounds JRW-0013, JRW-0051, JRW-0147, and JRW-0187 for 2 hours. 10 μM flimazine was added and BRET ratio 610/450 nm was measured. If the inhibitor, eg, JRW-0013, was permeable, the BRET ratio remained constant throughout the compound dose response curve. Intracellular BRET ratio was enhanced throughout the compound dose response curve while inhibiting spurious extracellular Nluc signal when the inhibitor, eg, JRW-0051, JRW-0147, or JRW-0187, was impermeable . See Figures 13B-13F.

Example 91
Cell Permeability by Bioluminescence Imaging HeLa cells were transiently transfected with a β2 adrenergic receptor-Nluc (B2AR-Nluc; C-terminal Nluc) or Nluc-β2 adrenergic receptor (Nluc-B2AR; N-terminal Nluc) fusion protein , And Nluc were fixed inside and outside of cells respectively and oriented. Twenty four hours after transfection, cells were treated with-/ + 30 μM thienopyrrole compounds JRW-0147, JRW-0051, and JRW-0138. 10 μM flimazine was added and luminescence was detected by imaging on an Olympus LV200. The thienopyrrole compounds JRW-0147 and JRW-0051 were impermeable, inhibited extracellular Nluc and enhanced intracellular Nluc. Compound JRW-0138 was cell permeable and inhibited both intracellular and extracellular Nluc. See FIG.

Example 92
Characterization of the Impermeable Thienopyrrole Compounds in the Target Association Model HEK 293 cells were transiently transfected with Src-Nluc fusion protein. See Figure 15A. Twenty-four hours after transfection, cells were treated with − / + 50 μg / mL digitonin to simulate the live and lytic scenarios, respectively. Intact and permeabilized cells were labeled with 1 μM dasatinib-DY605 tracer (impermeable) and treated with cell impermeable thienopyrrole compound JRW-0147 response curve for 2 hours. 10 μM frimazine was added and a BRET ratio of 610/450 nm was recorded. Figures 15B-15C show that JRW-0147 inhibited BRET in cell debris but not BRET inside cells.

Example 93
Time Course of Cell Permeability The following example demonstrates the permeability of the thienopyrrole compounds described herein. HEK 293 cells were transiently transfected with Nluc luciferase and expressed cytoplasmically. Twenty-four hours after transfection, cells were exposed to the compound response curve of the thienopyrrole compound JRW-0147 or JRW-0013 for 10 minutes, 30 minutes, or 120 minutes. Thereafter, 10 μM flimazine was added and luminescence was measured.

  If the thienopyrrole compound (inhibitor) is permeable, the compound enters the cells passively and dose dependent RLU from Nluc independently of time, as seen by JRW- 0013 (Figure 16) Decrease. If the thienopyrrole compound (inhibitor) is impermeable, the compound does not enter the cell actively or passively and does not depend on time, as seen by JRW-0147 (FIG. 16). No significant change of RLU from Nluc was observed.

Example 94
Measurement of endocytosis by chemical conjugation or molecular fusion to Nluc Antibodies, proteins, receptors, drugs, drug carriers, peptides, saccharides, fatty acids, nanoparticles, or other biomolecules are chemically conjugated to Nluc Once incorporated or fused, endocytosis could be measured in combination with the cell impermeable thienopyrrole compounds (inhibitors) described herein.

  For example, monoclonal antibodies (eg, Nluc-trastuzumab) could be chemically conjugated and bound to the HER2 receptor expressed on the surface of SKBR3 cells. A cell impermeable Nluc inhibitor could be applied to inhibit extracellular Nluc-trastuzumab. Active / passive internalized trastuzumab-Nluc-HER2 receptor (other antibodies chemically conjugated to Nluc, receptor, receptor, using gain of signal assay upon addition of coelenterazine substrate , Drugs, drug carriers, peptides, saccharides, fatty acids, nanoparticles, or other biomolecules could be measured kinetically.

  In another example, Nluc-GPCR (eg, Nluc-B2AR) was able to be genetically fused and expressed in mammalian cells. The cell impermeable thienopyrrole compounds (inhibitors) described herein could be applied to inhibit extracellular or membrane bound Nluc-B2AR. At the time of addition of the coelenterazine substrate, the gain of the signal assay can be used to extend to active / passive internalized or regenerated Nluc-GPCR (other proteins or receptors genetically fused to Nluc ) Could be measured kinetically.

Example 95
Specificity of the Inhibition of Nluc Luciferase The following example demonstrates Nluc luciferase activity and firefly luciferase activity, such as thienopyrrole compounds JRW-0251, JRW-0344, and JRW- of the present disclosure for inhibiting ULTRA GLO® luciferase. The specificity of 0147 is described. For Corning 3570 assay plates, a solution containing 1 μM luciferin in luciferase detection reagent (Promega Corporation V865 / 859) was added to the assay wells. Thereafter, equal volume titrations of the thienopyrrole compounds JRW-0251, JRW-0344, and JRW-0147 were added to the wells. Reactions were incubated for 2 hours at room temperature and luminescence was measured on a Tecan M1000 Pro plate reader. Table 3 shows that the thienopyrrole compounds did not inhibit firefly luciferase activity.
Table 3

Example 96
Thienopyrrole Compounds that Enable Multiplexing The following example describes the use of thienopyrrole compounds of the present invention to enable multiplexing of an assay that utilizes Nluc luciferase and another luciferase, such as firefly luciferase.

  For Corning 3570 assay plates, 1 × REAL-TIME-GLOTM MT Cell Viability Assay Reagent was prepared in 40 μL DMEM medium (n = 3) and incubated overnight. Next, a 40 μL titration of JRW-0147 or JRW-0344 (2 × concentration in CASPASE-GLO® 3/7 assay reagent (Promega Corporation)) was added. The reaction was incubated at room temperature and luminescence was judged at 1 hour.

Table 4 shows that the compounds inhibit background luminescence from the REALTIME-GLOTM assay in multiplexing with the CASPASE-GLOTM assay (medium containing the REALTIME-GLOTM reagent) Show. The compounds inhibit the Nluc enzyme and reduce background luminescence from the REALTIME-GLOTM reagent.
Table 4

Example 97
Cell Permeability The following example demonstrates the permeability of the thienopyrrole compounds described herein. HEK293 was transiently transfected with a β2 adrenergic receptor-Nluc (B2AR-Nluc) fusion protein to cytoplasmically fix and orient Nluc and pGEM-3z carrier DNA (1: 100). Twenty-four hours after transfection, cells were treated with − / + 50 μg / mL digitonin to mimic the live and lytic scenarios, respectively. The intact and permeabilized cells were then exposed to the compound response curve of the thienopyrrole compound for up to 2 hours. 10 μM flimazine (Promega Corp.) was added and luminescence was measured. When thienopyrrole compounds (inhibitors) were permeable, both live and permeable cell dose response curves overlapped. However, when the thienopyrrole compound was impermeable, EC50 was shifted to the right in living cells compared to permeable cells. 17A-17C show that JRW-0147 and JRW-0344 are cell impermeable characterized by EC50 shifted to the right in live vs. lysed cells. JRW-0013 served as a cell permeability control characterized by a similar EC50 in live versus lysed cells.

Example 98
Cell Viability and Toxicity The following example shows cell viability and toxicity of JRW-0344. HEK 293 cells were plated at 20 k cells / well and exposed to vehicle (DMSO), digitonin (positive control for cell death), or JRW-0344 for 10 min / 30 min / 120 min / 240 min / or 18 h. Cells Titer-Glo (Promega Corp.) were added and luminescence was measured. Figures 18A-18C show that after 18 hours there was no apparent toxicity of vehicle or JRW-0344 to 100 μM. Digitonin treated cells experienced almost complete death at concentrations above 2 μg / mL.

Example 99
Cell Permeability by Bioluminescence Imaging HeLa cells were transiently transfected with Nluc-ADRB2 (extracellular Nluc) or ADRB2-Nluc (intracellular Nluc), and Nluc was immobilized intracellularly or extracellularly and oriented. . Twenty-four hours after transfection, cells were treated with-/ + 30 μM thienopyrrole compounds JRW-0147, JRW-0013, and JRW-0344. 10 μM flimazine was added and luminescence was detected by imaging on an Olympus LV200. DMSO (untreated) was used as a negative control and did not inhibit Nluc in either orientation. JRW- 0013 is a cell permeable positive control and inhibited Nluc in either orientation. However, JRW-0344 or JRW-0147 inhibits the typical cyclic structure when Nluc is oriented extracellularly but not when Nluc is oriented intracellularly. See FIG.

Example 100
Inhibition of Extracellular Luciferase Activity The following example demonstrates the ability of the thienopyrrole compounds described herein to inhibit extracellular luciferase activity while enhancing intracellular BRET. See Figure 20A. HEK293 was transiently transfected with either SRC-Nluc or Nluc-HaloTag. Twenty-four hours after transfection, 20 k / cell was plated in the wells of a 96 well plate (Costar 3600). 1 μM cell impermeable tracer dasatinib-DY605 was added to HEK293 SRC-Nluc expressing cells, and 100 nM cell permeable tracer NanoBRET-618 was added to Nluc-HaloTag expressing cells. Both HEK293 SRC-Nluc and Nluc-HaloTag cells were exposed to the JRW-0344 dose response curve. FIGS. 20A-20C show that JRW-0344 inhibited BRET in cell debris in HEK293 SRC-Nluc expressing cells but not in HEK293 Nluc-HaloTag expressing cells.

Example 101
Inhibitor IC50 Determination The following example provides the IC50 values of the compounds disclosed herein. See Table 5. The NANOLUC® enzyme was diluted to 0.4 ng / ml in CO 2 independent medium + 10% FBS to make a detection reagent. A 3x dilution series of each inhibitor was then made with the detection reagent. A "no inhibitor" control was also generated for each sample. Fifty ul of each inhibitor dilution was mixed with 50 ul of NanoGlo buffer containing 20 uM frimazine (final flimazine concentration is 10 uM in Km) and luminescence was measured. Each sample was normalized to a "no inhibitor" control. IC50 values were then determined using GraphPad Prism (log [inhibitor] versus normalized response).
Table 5

  It is to be understood that the above detailed description and the appended examples are merely illustrative and do not limit the scope of the present invention which is defined solely by the appended claims and their equivalents.

  Various modifications and variations of the disclosed embodiments will be apparent to those skilled in the art. Such alterations and modifications including, but not limited to, chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations or methods of use of the invention are within the spirit and scope of the invention. You may go without deviating from.

  For the sake of completeness, various aspects of the present disclosure are presented in the following numbered clauses:

Clause 1. A compound of formula (I) or a salt thereof:
(I)
During the ceremony:
The dotted lines indicate the presence or absence of a bond;
n is 0, 1, 2, 3, 4 or 5;
X is CH, N, O, or S;
When the dotted line represents the presence of a bond, X is CH or N, and when the dotted line represents the absence of a bond, X is O or S;
A is an optionally substituted phenyl ring or an optionally substituted 5 or 6 membered heteroaryl ring;
R ¹ and R ² are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally And R ³ and R ⁴ are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally selected from the group consisting of: alkoxyalkyl substituted with and optionally substituted alkoxyalkoxyalkyl; And C ₃ -C ₈ -cycloalkyl, optionally substituted C ₃ -C ₈ -cycloalkylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl , Optionally selected from the group consisting of optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, and optionally substituted heterocyclylalkyl Or R ³ and R ⁴ together with the nitrogen atom to which they are attached form together an optionally substituted ring, a compound, or a salt thereof.

  Clause 2. The compound according to clause 1, wherein n is 1.

  Clause 3. The compound according to any one of the clauses 1-2, wherein said dotted line represents the presence of a bond and X is CH.

  Clause 4. 4. The compound according to any one of clauses 1 to 3, wherein A is a 5-membered heteroaryl ring.

  Clause 5. 5. The compound according to any one of paragraphs 1 to 4, wherein A is a thienyl ring or a furanyl ring.

  Clause 6. The compound according to any one of clauses 1-4, wherein A is a phenyl ring.

Clause 7. 7. The compound according to any one of clauses 1 to 6, wherein R ¹ is selected from the group consisting of hydrogen, C ₁ -C ₈ alkyl, halo-C ₁ -C ₈ -alkyl, alkoxyalkoxyalkyl and arylalkyl. Compound.

Clause 8. The compound according to any one of clauses 1 to 7, wherein R ¹ is selected from the group consisting of hydrogen, ethyl, n-hexyl, 2- (2-methoxyethoxy) ethyl and benzyl.

Clause 9. The compound according to any one of clauses 1-8, wherein R ¹ is ethyl.

Clause 10. 10. The compound according to any one of clauses 1-9, wherein R ² is optionally substituted aryl.

Clause 11. The compound according to any one of clauses 1 to 10, wherein R ² is substituted phenyl.

Clause 12. R ² is, C ₁ -C ₄ alkyl, cyano, amido, C ₁ -C ₄ alkoxy, and is phenyl substituted with one substituent selected from the group consisting of hydroxyalkyl, The compound according to clause 11.

Clause 13. The compound according to any one of the clauses 1-12, wherein R ² is phenyl substituted with one methyl group.

Clause 14. A compound according to any one of clauses 1 to 13, wherein R ³ and R ⁴ together with the nitrogen atom to which they are attached form together an optionally substituted ring.

Clause 15. The compound according to any one of the clauses 1-14, wherein R ³ and R ⁴ together with the nitrogen atom to which they are attached form together an optionally substituted monocyclic heterocycle.

  Clause 16. 25. The compound according to any one of the clauses 1-15, wherein the optionally substituted monocyclic heterocycle is selected from the group consisting of optionally substituted pyrrolidine, piperidine and piperazine.

  Clause 17. Wherein the optionally substituted monocyclic heterocycle is selected from the group consisting of unsubstituted pyrrolidine, unsubstituted piperidine, piperidine substituted with one substituent, or piperazine substituted with one substituent The compound of any one of 1 to 16.

Clause 18. 26. The compound according to any one of clauses 1-17, wherein R ³ is hydrogen.

Clause 19. R ⁴ is unsubstituted C ₁ -C ₈ alkyl, carboxy-C ₁ -C ₈ -alkyl, C ₁ -C ₄ -alkoxycarbonyl-C ₁ -C ₈ -alkyl, optionally substituted phenyl, optionally substituted C ₅ -C ₆ cycloalkyl ,, optionally substituted heterocyclyl, optionally substituted heteroaryl alkyl, and is selected from the group consisting of heterocyclylalkyl is optionally substituted, any provision 1-18 Or a compound according to item 1.

Clause 20. 22. The compound according to any one of clauses 1-19, wherein R ⁴ is phenyl which is unsubstituted or substituted with one substituent.

Clause 21. Substituents, C ₁ -C ₄ - alkoxycarbonyl, and C ₁ -C ₄ - alkoxycarbonyl -C ₁ -C ₄ - is selected from the group consisting of alkyl, The compound according to clause 20.

Clause 22. R ⁴ is carboxy, C ₁ -C ₄ - alkoxycarbonyl, C ₁ -C ₈ - alkyl amide, hydroxy -C ₁ -C ₈ - alkyl, amide, amino -C substituted with optionally ₁ -C ₈ - Alkylamide, C ₁ -C ₄ -dialkylamino-C ₁ -C ₈ -alkylamide, carboxy-C ₁ -C ₈ -alkylamide, sulfonic acid -C ₁ -C ₈ -alkylamide, sulfonic acid -C _1- C ₈ - alkyl amide, C ₁ -C ₄ - alkylcarbonyl -C ₁ -C ₈ - alkyl amide, C ₃ -C ₆ substituted by optionally - cycloalkyl amides, and the group consisting of heterocyclylalkyl amide substituted with optionally 22. A compound according to any one of the clauses 1-18, which is cyclohexyl substituted with one substituent selected from

Clause 23. The compound is of the formula (Ia):
(Ia)
The compound according to any one of the clauses 1-22, having a.

Clause 24. The compound has the formula (Ib):
(Ib)
During the ceremony:
Y is selected from the group consisting of -NR ^a R ^b and -OR ^c ;
R ^a and R ^b are each independently hydrogen, C substituted with optionally ₁ -C ₈ alkyl, C ₃ -C ₈ substituted with optionally - cycloalkyl, the group consisting of heterocyclyl substituted with optionally Or R ^a and R ^b together with the nitrogen atom to which they are attached together form an optionally substituted ring; and R ^c is hydrogen and optionally substituted C ₁ It is selected from the group consisting of -C ₄ alkyl, a compound according to any one of clauses 1-23.

Clause 25. 26. The compound according to clause 24, wherein Y is -OR ^c .

Clause 26. 26. The compound according to any one of clauses 24-25, wherein R ^<c > is selected from the group consisting of hydrogen and methyl.

Clause 27. Y is -NR ^a R ^b, a compound according to clause 24.

Clause 28. 26. The compound according to clause 27, wherein R ^a and R ^b together with the nitrogen atom to which they are attached form an optionally substituted ring.

Clause 29. 29. The compound according to any one of the clauses 27-28, wherein R ^a and R ^b together with the nitrogen atom to which they are attached form an optionally substituted monocyclic heterocycle.

  Clause 30. 30. The compound according to clause 29, wherein said optionally substituted monocyclic heterocycle is optionally substituted piperidine.

  Clause 31. 31. The compound according to any one of clauses 29 to 30, wherein the optionally substituted monocyclic heterocycle is selected from the group consisting of unsubstituted piperidine and piperidine substituted with one substituent.

Clause 32. 26. The compound according to clause 27, wherein R ^a is hydrogen.

Clause 33. Clause 32 wherein R ^b is selected from the group consisting of hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally substituted C ₃ -C ₈ -cycloalkyl, and optionally substituted heterocyclyl Compound described.

Clause 34. R ^b is hydrogen, C ₁ -C ₆ alkyl, hydroxyalkyl, optionally substituted aminoalkyl, carboxyalkyl, sulfonic acid-alkyl, sulfonate-alkyl, alkylcarbonylalkyl, optionally substituted C ₃ -C ₆ 43. A compound according to any one of clauses 31 to 32, selected from the group consisting of-cycloalkyl and optionally substituted 6 membered heterocyclyl.

Clause 35. Said compound has the following formula (Ib ')
(Ib ')
The compound of any one of clauses 1-34, having

Clause 36. The compound is
N-Cyclohexyl-4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;
N-ethyl-2- (5- (pyrrolidine-1-carbonyl) -4H-thieno [3,2-b] pyrrol-4-yl) -N- (m-tolyl) acetamide;
N-ethyl-2- (5- (piperidine-1-carbonyl) -4H-thieno [3,2-b] pyrrol-4-yl) -N- (m-tolyl) acetamide;
1- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carbonyl) piperidine-4-carboxylate;
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N-phenyl-4H-thieno [3,2-b] pyrrole-5-carboxamide;
2- (4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) phenyl) ethyl acetate;
Methyl 3- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) benzoate;
Methyl-cis-4- (4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid;
8- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) octanoic acid;
6- (4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) piperidin-1-yl) -6 -Oxohexanoic acid;
Trans-methyl-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid;
Trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid;
N- (trans-4- (butylcarbamoyl) cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (trans-4-((2-hydroxyethyl) carbamoyl) cyclohexyl) -4H-thieno [3,2-b] pyrrole -5- carboxamide;
N- (trans-4-((2- (dimethylamino) ethyl) carbamoyl) cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2- b] pyrrole-5-carboxamide;
4- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1-carboxamide) Butanoic acid;
N- (trans-4-carbamoylcyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (trans-4- (hexylcarbamoyl) cyclohexyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;
1- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carbonyl) Ethyl piperidine-4-carboxylate;
6- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxamide) Methyl hexanoate;
6- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxamide) Hexanoic acid;
1- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carbonyl) Piperidine-4-carboxylic acid;
8- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxamide) Octanoic acid;
N- (trans-4- (cyclohexylcarbamoyl) cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (trans-4-((1-methylpiperidin-4-yl) carbamoyl) cyclohexyl) -4H-thieno [3,2 -B] pyrrole-5-carboxamide;
4- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1-carboxamide) Piperidine-1-carboxylic acid tert-butyl;
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (trans-4- (piperidin-4-ylcarbamoyl) cyclohexyl) -4H-thieno [3,2-b] pyrrole- 5-carboxamide;
N- (trans-4-((1-acetylpiperidin-4-yl) carbamoyl) cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2 -B] pyrrole-5-carboxamide;
(6- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1-carboxamide ) Hexyl) tert-butyl carbamate;
N- (trans-4-((6- (3 ′, 6′-dihydroxy-3-oxo-3H-spiro [isobenzofuran-1,9′-xanthene] -5 (6) -carboxamido) hexyl) carbamoyl) Cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;
N- (trans-4-((6-aminohexyl) carbamoyl) cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole -5-carboxamide hydrochloride;
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (trans-4-((6-hydroxyhexyl) carbamoyl) cyclohexyl) -4H-thieno [3,2-b] pyrrole -5- carboxamide;
Methyl-trans-4- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane 1-carboxamide) cyclohexane-1-carboxylic acid;
Trans-4- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1- Carboxamide) cyclohexane-1-carboxylic acid;
(11S, 14S, 17S) -17-acetamido-11, 14-bis (carboxymethyl) -1- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl)- 4 H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexyl) -1,10,13,16-tetraoxo-2,9,12,15-tetraazanonadecane-19-oic acid;
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N-methyl-4H-thieno [3,2-b] pyrrole-5-carboxamide;
N-Cyclopentyl-4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (pyridin-4-ylmethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (3-morpholinopropyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;
N-ethyl-2- (5- (4-methylpiperazine-1-carbonyl) -4H-thieno [3,2-b] pyrrol-4-yl) -N- (m-tolyl) acetamide;
Methyl 4- (2-oxo-2- (m-tolylamino) ethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate;
N-Cyclohexyl-4- (2-oxo-2- (m-tolylamino) ethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;
N-Cyclohexyl-4- (2- (2- (2- (2-methoxyethoxy) ethyl) (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;
Methyl-trans-4- (4- (2- (2- (2-methoxyethoxy) ethyl) (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5 -Carboxamide) cyclohexane 1-carboxylic acid;
4- (2- (Hexyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate methyl;
N-cyclohexyl-4- (2- (hexyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;
Methyl-trans-4- (4- (2- (hexyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid;
4- (2- (benzyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate methyl;
6- (cis-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxamide) Hexanoic acid;
6- (trans-4-((4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) methyl) cyclohexane-1 -Carboxamide) methyl hexanoic acid;
6- (trans-4-((4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) methyl) cyclohexane-1 -Carboxamide) hexanoic acid;
6- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxamide) Sodium hexane-1-sulfonate;
6- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxamide) Hexane-1-sulfonic acid potassium;
Trans-4- (4- (2- (Hexyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1-carboxylic acid;
Trans-4- (4- (2- (ethyl (phenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid methyl;
Trans-4- (4- (2-((3-Cyanophenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid Methyl;
Trans-4- (4- (2-((3-carbamoylphenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid Methyl;
Trans-4- (4- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid methyl;
Trans-4- (4- (2- (ethyl (3-methoxyphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid methyl;
Trans-4- (4- (2- (ethyl (o-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid methyl;
Trans-4- (4- (2- (6-methyl-3,4-dihydroquinolin-1 (2H) -yl) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide ) Methyl cyclohexane-1 -carboxylate;
Trans-4- (4- (2- (ethyl (p-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid methyl;
Trans-4- (4- (2- (ethyl (4- (hydroxymethyl) phenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carvone Methyl acid;
Trans-4- (1- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -1H-indole-2-carboxamide) methyl cyclohexane-1-carboxylate;
Trans-4- (1- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -6-methoxy-1H-indole-2-carboxamido) cyclohexane-1-carboxylate methyl;
Trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-furo [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid methyl;
4- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -N- (trans-4-((6-hydroxyhexyl) carbamoyl) cyclohexyl) -4H-thieno [3,2-b] Pyrrole-5-carboxamide;
6- (trans-4- (4- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1-carboxamide ) Hexane-1-sulfonic acid sodium;
Trans-4- (4- (2- (ethyl (3-isopropylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid methyl;
Trans-4- (4- (2- (ethyl (3- (hydroxymethyl) phenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid Methyl acid;
Trans-4- (4- (2-((3- (bromomethyl) phenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1- Methyl carboxylate;
Trans-4- (4- (2-((3- (dimethylamino) phenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1 -Methyl carboxylate;
Trans-4- (4- (2- (ethyl (3-isobutylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid methyl;
Trans-4- (1- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -1H-indole-2-carboxamide) methyl cyclohexane-1-carboxylate;
1- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -N- (trans-4-((6-hydroxyhexyl) carbamoyl) cyclohexyl) -1H-indole-2-carboxamide;
6- (trans-4- (1- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -1H-indole-2-carboxamido) cyclohexane-1-carboxamido) hexane-1-sulfonic acid sodium ;
Trans-4- (1- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -1H-pyrrole-2-carboxamide) methyl cyclohexane-1-carboxylate; Clause 1 selected from the group consisting of methyl 2- (ethyl (m-tolyl) amino) -2-oxoethyl) -6H-thieno [2,3-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylate The compound of any one of -35.

  Clause 37. A method for inhibiting Oplophorus-derived luciferase, comprising contacting Oplophorus-derived luciferase with the compound according to any one of Articles 1-36.

  Clause 38. 37. The method according to clause 37, wherein the Oplophorus derived luciferase comprises the polypeptide sequence of SEQ ID NO: 2.

Clause 39. A method for inhibiting Oplophorus-derived luciferase, comprising
The Oplophorus-derived luciferase is a compound of the formula (II):
(II)
Including contacting with
During the ceremony:
The dotted lines indicate the presence or absence of a bond;
n is 0, 1, 2, 3, 4 or 5;
X is CH, N, O, or S;
When X represents CH or N when the dotted line represents the presence of a bond, X is O or S when the dotted line represents the absence of a bond;
A is an optionally substituted phenyl ring, or an optionally substituted 5 or 6 membered heteroaryl ring;
R ¹ and R ² are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally Selected from the group consisting of alkoxyalkyl substituted with and optionally substituted alkoxyalkoxyalkyl;
Z is selected from the group consisting of -NR ³ R ⁴ and -OR ⁵ ; and R ³ , R ⁴ and R ⁵ are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, Optionally substituted C ₃ -C ₈ -cycloalkyl, optionally substituted C ₃ -C ₈ -cycloalkylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted hetero Selected from the group consisting of aryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, and optionally substituted heterocyclylalkyl; or R ³ and R ⁴ with the nitrogen atom to which they are attached Together, optionally together forming a ring.

  Clause 40. A method according to clause 39, wherein the Oplophorus derived luciferase comprises the polypeptide sequence of SEQ ID NO: 2.

Clause 41. A method of controlling luminescence of Oplophorus-derived luciferase in a sample, comprising
(A) contacting the sample with a coelenterazine substrate and a compound according to any one of clauses 1 to 36; and (b) detecting luminescence in the sample,
Including
The method wherein the compound according to any one of the clauses 1-36 causes a decrease in luminescence from the Oplophorus derived luciferase.

Clause 42. A method of detecting an interaction between a first protein and a second protein in a sample comprising:
(A) contacting the sample with a coelenterazine substrate and a compound according to any one of clauses 1 to 36, wherein the sample is
(Ix) a first polynucleotide encoding a first fusion protein comprising a first fragment of the Oplophorus-derived luciferase and a first protein; and (x) a second fragment and a second fragment of the Oplophorus-derived luciferase A second polynucleotide encoding a second fusion protein comprising the protein; and (b) detecting luminescence in the sample,
Including
The method wherein the detection of luminescence indicates an interaction between the first protein and the second protein.

  Clause 43. The method according to any one of clauses 41-42, comprising contacting the sample with a coelenterazine substrate prior to contacting the sample with a compound according to any one of clauses 1-36.

  Clause 44. When the first protein and the second protein interact with each other, a full-length enzyme capable of stably binding the first fragment of the Oplophorus-derived luciferase and the second fragment of the Oplophorus-derived luciferase to a coelenterazine substrate 42. The method according to clause 42, to reconstitute.

Clause 45. A method of detecting an interaction between a first protein and a second protein in a sample comprising:
(A) contacting the sample with a coelenterazine substrate and a compound according to any one of clauses 1-36, wherein the sample is:
(Xi) a first polynucleotide encoding a first fusion protein comprising a bioluminescent donor Oplophorus-derived luciferase and a first protein; and (xii) a second fusion comprising a fluorescent acceptor molecule and a second protein Comprising a second polynucleotide encoding a protein;
(B) detecting bioluminescence resonance energy transfer (BRET) in a sample indicative of the interaction or proximity of the bioluminescence donor and the fluorescence acceptor.

  Clause 46. 42. The method of any one of clauses 41-45, wherein the sample comprises cells.

  Clause 47. 51. The method of clause 46, wherein the cells comprise the Oplophorus derived luciferase.

  Clause 48. The method according to paragraph 46, wherein the cells express the Oplophorus derived luciferase.

  Clause 49. 43. The method according to any one of clauses 41 to 48, wherein the coelenterazine substrate is coelenterazine, coelenterazine derivative, coelenterazine analogue, procoelenterazine, or quinone masked coelenterazine.

  Clause 50. A first fusion protein comprising a first target protein and a bioluminescent donor molecule, wherein the bioluminescent donor molecule is an Oplophorus-derived luciferase; a second fusion protein comprising a second target protein and a fluorescent acceptor molecule; A bioluminescence resonance energy transfer (BRET) system comprising a coelenterazine substrate, and a compound according to any of the clauses 1-36.

Clause 51. A kit comprising: (a) the compound according to any one of paragraphs 1 to 36; and (b) an Oplophorus-derived luciferase.

  Clause 52. The kit of clause 51, wherein the Oplophorus-derived luciferase comprises the polypeptide sequence of SEQ ID NO: 2.

  Clause 53. 51. The kit of any one of clauses 51-52, further comprising a coelenterazine substrate.

  Clause 54. 61. The kit of any one of clauses 51-53, further comprising instructions for performing a luminescence assay.

Clause 55. A method of controlling luminescence of Oplophorus-derived luciferase in a sample, comprising
(A) contacting the sample with coelenterazine substrate and a compound of formula (II):
(II)
Including
During the ceremony:
The dotted lines indicate the presence or absence of a bond;
n is 0, 1, 2, 3, 4 or 5;
X is CH, N, O, or S;
When X represents CH or N when the dotted line represents the presence of a bond, X is O or S when the dotted line represents the absence of a bond;
A is an optionally substituted phenyl ring, or an optionally substituted 5 or 6 membered heteroaryl ring;
R ¹ and R ² are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally Selected from the group consisting of alkoxyalkyl substituted with and optionally substituted alkoxyalkoxyalkyl;
Z is selected from the group consisting of -NR ³ R ⁴ and -OR ⁵ ; and R ³ , R ⁴ and R ⁵ are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, Optionally substituted C ₃ -C ₈ -cycloalkyl, optionally substituted C ₃ -C ₈ -cycloalkylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted hetero Selected from the group consisting of aryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, and optionally substituted heterocyclylalkyl; or R ³ and R ⁴ with the nitrogen atom to which they are attached Together forming an optionally substituted ring together; and (b) detecting luminescence in the sample,
The method wherein the compound of formula (II) causes a decrease in luminescence from an Oplophorus derived luciferase.

Clause 56. A method of detecting an interaction between a first protein and a second protein in a sample comprising:
(A) contacting the sample with coelenterazine substrate and a compound of formula (II),
(II)
During the ceremony:
The dotted lines indicate the presence or absence of a bond;
n is 0, 1, 2, 3, 4 or 5;
X is CH, N, O, or S;
When X represents CH or N when the dotted line represents the presence of a bond, X is O or S when the dotted line represents the absence of a bond;
A is an optionally substituted phenyl ring, or an optionally substituted 5 or 6 membered heteroaryl ring;
R ¹ and R ² are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally Selected from the group consisting of alkoxyalkyl substituted with and optionally substituted alkoxyalkoxyalkyl;
Z is selected from the group consisting of -NR ³ R ⁴ and -OR ⁵ ; and R ³ , R ⁴ and R ⁵ are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, Optionally substituted C ₃ -C ₈ -cycloalkyl, optionally substituted C ₃ -C ₈ -cycloalkylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted hetero Selected from the group consisting of aryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, and optionally substituted heterocyclylalkyl; or R ³ and R ⁴ with the nitrogen atom to which they are attached Together, form an optionally substituted ring together,
The sample is
(Xiii) a first polynucleotide encoding a first fusion protein comprising a first fragment of Oplophorus-derived luciferase and a first protein; and (xiv) a second fragment of the Oplophorus-derived luciferase and a second protein A second polynucleotide encoding a second fusion protein; and (b) detecting luminescence in said sample,
The method wherein the detection of luminescence indicates an interaction between the first protein and the second protein.

  Clause 57. 61. A method according to any one of clauses 55 to 56, comprising contacting the sample with coelenterazine substrate prior to contacting the sample with a compound of formula (II).

  Clause 58. When the first protein and the second protein interact with each other, the first fragment of the Oplophorus-derived luciferase and the second fragment of the Oplophorus-derived luciferase are stably bound to a coelenterazine substrate 57. A method according to clause 57, which reconstitutes a possible full-length enzyme.

Clause 59. A method of detecting an interaction between a first protein and a second protein in a sample comprising:
(A) contacting the sample with coelenterazine substrate and a compound of formula (II),
(II)
During the ceremony:
The dotted lines indicate the presence or absence of a bond;
n is 0, 1, 2, 3, 4 or 5;
X is CH, N, O, or S;
When X represents CH or N when the dotted line represents the presence of a bond, X is O or S when the dotted line represents the absence of a bond;
A is an optionally substituted phenyl ring, or an optionally substituted 5 or 6 membered heteroaryl ring;
R ¹ and R ² are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally Selected from the group consisting of alkoxyalkyl substituted with and optionally substituted alkoxyalkoxyalkyl;
Z is selected from the group consisting of -NR ³ R ⁴ and -OR ⁵ ; and R ³ , R ⁴ and R ⁵ are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, Optionally substituted C ₃ -C ₈ -cycloalkyl, optionally substituted C ₃ -C ₈ -cycloalkylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted hetero Selected from the group consisting of aryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, and optionally substituted heterocyclylalkyl; or R ³ and R ⁴ with the nitrogen atom to which they are attached Together, form an optionally substituted ring together,
The sample is
(Xv) a first polynucleotide encoding a first fusion protein comprising a bioluminescent donor Oplophorus-derived luciferase and a first protein; and (xvi) a second fusion comprising a fluorescent acceptor molecule and a second protein (B) detecting bioluminescence resonance energy transfer (BRET) in the sample, which comprises: a second polynucleotide encoding a protein; and (b) indicating the interaction or proximity of the bioluminescence donor and the fluorescence acceptor. , Said method.

  Clause 60. 61. The method of any one of clauses 55-59, wherein the sample comprises cells.

  Clause 61. 61. The method according to clause 60, wherein the cells comprise the Oplophorus derived luciferase.

  Clause 62. 61. The method according to clause 60, wherein the cells express the Oplophorus derived luciferase.

  Clause 63. The method according to any one of paragraphs 54 to 62, wherein the coelenterazine substrate is coelenterazine, coelenterazine derivative, coelenterazine analogue, procoelenterazine, or quinone masked coelenterazine.

Clause 64. A bioluminescent resonance energy transfer (BRET) system,
A first fusion protein comprising a first target protein and a bioluminescent donor molecule, wherein said bioluminescent donor molecule is an Oplophorus-derived luciferase; a second fusion protein comprising a second target protein and a fluorescent acceptor molecule; A coelenterazine substrate, and a compound of formula (II),
(II)
During the ceremony:
The dotted lines indicate the presence or absence of a bond;
n is 0, 1, 2, 3, 4 or 5;
X is CH, N, O, or S;
When X represents CH or N when the dotted line represents the presence of a bond, X is O or S when the dotted line represents the absence of a bond;
A is an optionally substituted phenyl ring, or an optionally substituted 5 or 6 membered heteroaryl ring;
R ¹ and R ² are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally Selected from the group consisting of alkoxyalkyl substituted with and optionally substituted alkoxyalkoxyalkyl;
Z is selected from the group consisting of -NR ³ R ⁴ and -OR ⁵ ; and R ³ , R ⁴ and R ⁵ are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, Optionally substituted C ₃ -C ₈ -cycloalkyl, optionally substituted C ₃ -C ₈ -cycloalkylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted hetero Selected from the group consisting of aryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, and optionally substituted heterocyclylalkyl; or R ³ and R ⁴ with the nitrogen atom to which they are attached A bioluminescent resonance energy transfer (BRET) system that together form an optionally substituted ring.

Clause 65. (A) Compound of Formula (II):
(II)
During the ceremony:
The dotted lines indicate the presence or absence of a bond;
n is 0, 1, 2, 3, 4 or 5;
X is CH, N, O, or S;
When X represents CH or N when the dotted line represents the presence of a bond, X is O or S when the dotted line represents the absence of a bond;
A is an optionally substituted phenyl ring, or an optionally substituted 5 or 6 membered heteroaryl ring;
R ¹ and R ² are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally Selected from the group consisting of alkoxyalkyl substituted with and optionally substituted alkoxyalkoxyalkyl;
Z is selected from the group consisting of -NR ³ R ⁴ and -OR ⁵ ; and R ³ , R ⁴ and R ⁵ are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, Optionally substituted C ₃ -C ₈ -cycloalkyl, optionally substituted C ₃ -C ₈ -cycloalkylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted hetero Selected from the group consisting of aryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, and optionally substituted heterocyclylalkyl; or R ³ and R ⁴ with the nitrogen atom to which they are attached Together, together form an optionally substituted ring; and (b) a luciferase comprising Oplophorus.

  Clause 66. The kit according to clause 65, wherein the Oplophorus derived luciferase comprises the polypeptide sequence of SEQ ID NO: 2.

  Clause 67. 65. The kit of any one of clauses 65-66, further comprising a coelenterazine substrate.

Clause 68. The kit according to any one of clauses 65 to 67, further comprising instructions for performing a luminescence assay.
Appendix

SEQ ID NO: 1-Natural mature Oplophorus luciferase amino acid sequence FTLADFVGDWQQTAGYNQDQVLEQGLSLFQALGSVSV TPIQKV VLSGENGLKADIH VII PYEGLSGFQ MGLEMIFKV VYPVDDHHFILGITGVTPNMIDYFGRPYPGI VFD GDQKQITTITGTGTGTGTGTGTGTGTGTGTG V TGL VIL

SEQ ID NO: 2-Nluc amino acid sequence MVFTLEDFVGD WRQ TAG YNLDQ VLEQGGVSSLFQNLGVSVTPIQRIVLS GENGLKIDIL VIIPYEGLSGDQ MGQ IEKIFKVV YPVDDHHFKVILHYGTV IDGVTPNMIDYEGIAVFDGKKITVGTLWNGNKILERPDGLL VIV

Claims (68)

A compound of formula (I) or a salt thereof:
(I)
During the ceremony:
The dotted lines indicate the presence or absence of a bond;
n is 0, 1, 2, 3, 4 or 5;
X is CH, N, O, or S;
When the dotted line represents the presence of a bond, X is CH or N, and when the dotted line represents the absence of a bond, X is O or S;
A is an optionally substituted phenyl ring or an optionally substituted 5 or 6 membered heteroaryl ring;
R ¹ and R ² are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally And R ³ and R ⁴ are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally selected from the group consisting of: alkoxyalkyl substituted with and optionally substituted alkoxyalkoxyalkyl; And C ₃ -C ₈ -cycloalkyl, optionally substituted C ₃ -C ₈ -cycloalkylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl , Optionally selected from the group consisting of optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, and optionally substituted heterocyclylalkyl Or R ³ and R ⁴ together with the nitrogen atom to which they are attached together form an optionally substituted ring, or a salt thereof.   The compound according to claim 1, wherein n is 1.   The compound according to any one of claims 1-2, wherein said dotted line represents the presence of a bond and X is CH.   The compound according to any one of claims 1 to 3, wherein A is a 5-membered heteroaryl ring.   The compound according to any one of claims 1 to 4, wherein A is a thienyl ring or a furanyl ring.   The compound according to any one of claims 1 to 3, wherein A is a phenyl ring. R ¹ is hydrogen, C ₁ -C ₈ alkyl, halo -C ₁ -C ₈ - alkyl, alkoxy alkoxyalkyl, and is selected from the group consisting of arylalkyl, according to any one of claims 1 to 6 Compounds. R ¹ is hydrogen, ethyl, n- hexyl, 2- (2-methoxyethoxy) is selected from the group consisting of ethyl and benzyl, A compound according to any one of claims 1 to 7. R ¹ is ethyl, A compound according to any one of claims 1 to 8. R ² is aryl substituted with optionally, a compound according to any one of claims 1-9. R ² is substituted phenyl, A compound according to claim 10. The compound according to claim 11, wherein R ² is phenyl substituted with one substituent selected from the group consisting of C ₁ -C ₄ alkyl, cyano, amido, C ₁ -C ₄ alkoxy and hydroxyalkyl. R ² is phenyl substituted with one methyl group, A compound according to claim 12. R ³ and R ^4, together with the nitrogen atom to which they are attached, together form a ring which is optionally substituted, a compound according to any one of claims 1 to 13. R ³ and R ^4, together with the nitrogen atom to which they are attached, form a heterocyclic ring of monocyclic substituted optionally with compound according to claim 14.   16. The compound according to claim 15, wherein the optionally substituted monocyclic heterocycle is selected from the group consisting of optionally substituted pyrrolidine, piperidine and piperazine.   The optionally substituted monocyclic heterocycle is selected from the group consisting of unsubstituted pyrrolidine, unsubstituted piperidine, piperidine substituted with one substituent, or piperazine substituted with one substituent. A compound according to claim 15. R ³ is hydrogen, A compound according to any one of claims 1-17. R ⁴ is unsubstituted C ₁ -C ₈ alkyl, halo-C ₁ -C ₈ -alkyl, carboxy-C ₁ -C ₈ -alkyl, C ₁ -C ₄ -alkoxycarbonyl-C ₁ -C ₈ -alkyl, optionally substituted phenyl, C ₅ -C ₆ cycloalkyl substituted with optionally, C ₅ -C an optionally substituted ₆ - cycloalkylalkyl, heterocyclyl optionally substituted, heteroarylalkyl which are optionally substituted 19. A compound according to any one of the preceding claims, which is selected from the group consisting of and optionally substituted heterocyclylalkyl. R ⁴ is phenyl, unsubstituted or substituted with one substituent group A compound according to claim 19. It said substituents, C ₁ -C ₄ - alkoxycarbonyl, and C ₁ -C ₄ - alkoxycarbonyl -C ₁ -C ₄ - is selected from the group consisting of alkyl, A compound according to claim 20. R ⁴ is carboxy, C ₁ -C ₄ - alkoxycarbonyl, C ₁ -C ₈ - alkyl amide, hydroxy -C ₁ -C ₈ - alkyl, amide, amino -C substituted with optionally ₁ -C ₈ - Alkylamide, C ₁ -C ₄ -dialkylamino-C ₁ -C ₈ -alkylamide, carboxy-C ₁ -C ₈ -alkylamide, sulfonic acid -C ₁ -C ₈ -alkylamide, sulfonic acid -C _1- C ₈ - alkyl amide, C ₁ -C ₄ - alkylcarbonyl -C ₁ -C ₈ - alkyl amide, C ₃ -C ₆ substituted by optionally - cycloalkyl amides, and the group consisting of heterocyclylalkyl amide substituted with optionally 19. The compound according to any one of claims 1-18, which is cyclohexyl substituted with one substituent selected from The compound is of the formula (Ia):
(Ia)

A compound according to claim 1 having Said compound has the formula (Ib):
(Ib)
During the ceremony:
Y is selected from the group consisting of -NR ^a R ^b and -OR ^c ;
R ^a and R ^b are each independently hydrogen, C substituted with optionally ₁ -C ₈ alkyl, C ₃ -C ₈ substituted with optionally - cycloalkyl, the group consisting of heterocyclyl substituted with optionally Or R ^a and R ^b together with the nitrogen atom to which they are attached together form an optionally substituted ring; and R ^c is hydrogen and optionally substituted C ₁ It is selected from the group consisting of -C ₄ alkyl, a compound according to claim 1. 25. The compound of claim 24, wherein Y is -OR ^c . 26. The compound of claim 25, wherein R ^c is selected from the group consisting of hydrogen and methyl. Y is -NR ^a R ^b, compound of claim 24. 28. The compound of claim 27, wherein R ^a and R ^b together with the nitrogen atom to which they are attached form an optionally substituted ring. R ^a and R ^b together with the nitrogen atom to which they are attached, form a heterocyclic ring of monocyclic substituted optionally with compound according to claim 28.   30. The compound of claim 29, wherein the optionally substituted monocyclic heterocycle is an optionally substituted piperidine.   31. The compound according to claim 30, wherein the optionally substituted monocyclic heterocycle is selected from the group consisting of unsubstituted piperidine and piperidine substituted with one substituent. 28. The compound of claim 27, wherein R ^a is hydrogen. 34. The method according to claim 32, wherein R ^b is selected from the group consisting of hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally substituted C ₃ -C ₈ -cycloalkyl, and optionally substituted heterocyclyl. The compound as described in. R ^b is hydrogen, C ₁ -C ₆ alkyl, hydroxyalkyl, optionally substituted aminoalkyl, carboxyalkyl, sulfonic acid-alkyl, sulfonate-alkyl, alkylcarbonylalkyl, optionally substituted C ₃ -C ₆ 33. A compound according to claim 32, selected from the group consisting of-cycloalkyl and optionally substituted 6 membered heterocyclyl. Said compound has the following formula (Ib ')
(Ib ')
35. A compound according to any one of claims 24 to 34, having the formula: The compound is
N-Cyclohexyl-4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;
N-ethyl-2- (5- (pyrrolidine-1-carbonyl) -4H-thieno [3,2-b] pyrrol-4-yl) -N- (m-tolyl) acetamide;
N-ethyl-2- (5- (piperidine-1-carbonyl) -4H-thieno [3,2-b] pyrrol-4-yl) -N- (m-tolyl) acetamide;
1- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carbonyl) piperidine-4-carboxylate;
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N-phenyl-4H-thieno [3,2-b] pyrrole-5-carboxamide;
2- (4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) phenyl) ethyl acetate;
Methyl 3- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) benzoate;
Methyl-cis-4- (4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid;
8- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) octanoic acid;
6- (4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) piperidin-1-yl) -6 -Oxohexanoic acid;
Trans-methyl-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid;
Trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid;
N- (trans-4- (butylcarbamoyl) cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (trans-4-((2-hydroxyethyl) carbamoyl) cyclohexyl) -4H-thieno [3,2-b] pyrrole -5- carboxamide;
N- (trans-4-((2- (dimethylamino) ethyl) carbamoyl) cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2- b] pyrrole-5-carboxamide;
4- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1-carboxamide) Butanoic acid;
N- (trans-4-carbamoylcyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (trans-4- (hexylcarbamoyl) cyclohexyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;
1- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carbonyl) Ethyl piperidine-4-carboxylate;
6- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxamide) Methyl hexanoate;
6- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxamide) Hexanoic acid;
1- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carbonyl) Piperidine-4-carboxylic acid;
8- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxamide) Octanoic acid;
N- (trans-4- (cyclohexylcarbamoyl) cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (trans-4-((1-methylpiperidin-4-yl) carbamoyl) cyclohexyl) -4H-thieno [3,2 -B] pyrrole-5-carboxamide;
4- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1-carboxamide) Piperidine-1-carboxylic acid tert-butyl;
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (trans-4- (piperidin-4-ylcarbamoyl) cyclohexyl) -4H-thieno [3,2-b] pyrrole- 5-carboxamide;
N- (trans-4-((1-acetylpiperidin-4-yl) carbamoyl) cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2 -B] pyrrole-5-carboxamide;
(6- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1-carboxamide ) Hexyl) tert-butyl carbamate;
N- (trans-4-((6- (3 ′, 6′-dihydroxy-3-oxo-3H-spiro [isobenzofuran-1,9′-xanthene] -5 (6) -carboxamido) hexyl) carbamoyl) Cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;
N- (trans-4-((6-aminohexyl) carbamoyl) cyclohexyl) -4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole -5-carboxamide hydrochloride;
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (trans-4-((6-hydroxyhexyl) carbamoyl) cyclohexyl) -4H-thieno [3,2-b] pyrrole -5- carboxamide;
Methyl-trans-4- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane 1-carboxamide) cyclohexane-1-carboxylic acid;
Trans-4- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1- Carboxamide) cyclohexane-1-carboxylic acid;
(11S, 14S, 17S) -17-acetamido-11, 14-bis (carboxymethyl) -1- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl)- 4 H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexyl) -1,10,13,16-tetraoxo-2,9,12,15-tetraazanonadecane-19-oic acid;
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N-methyl-4H-thieno [3,2-b] pyrrole-5-carboxamide;
N-Cyclopentyl-4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (pyridin-4-ylmethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;
4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -N- (3-morpholinopropyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;
N-ethyl-2- (5- (4-methylpiperazine-1-carbonyl) -4H-thieno [3,2-b] pyrrol-4-yl) -N- (m-tolyl) acetamide;
Methyl 4- (2-oxo-2- (m-tolylamino) ethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate;
N-Cyclohexyl-4- (2-oxo-2- (m-tolylamino) ethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;
N-Cyclohexyl-4- (2- (2- (2- (2-methoxyethoxy) ethyl) (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;
Methyl-trans-4- (4- (2- (2- (2-methoxyethoxy) ethyl) (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5 -Carboxamide) cyclohexane 1-carboxylic acid;
4- (2- (Hexyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate methyl;
N-cyclohexyl-4- (2- (hexyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide;
Methyl-trans-4- (4- (2- (hexyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid;
4- (2- (benzyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxylate methyl;
6- (cis-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxamide) Hexanoic acid;
6- (trans-4-((4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) methyl) cyclohexane-1 -Carboxamide) methyl hexanoic acid;
6- (trans-4-((4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) methyl) cyclohexane-1 -Carboxamide) hexanoic acid;
6- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxamide) Sodium hexane-1-sulfonate;
6- (trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxamide) Hexane-1-sulfonic acid potassium;
Trans-4- (4- (2- (Hexyl (m-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1-carboxylic acid;
Trans-4- (4- (2- (ethyl (phenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid methyl;
Trans-4- (4- (2-((3-Cyanophenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid Methyl;
Trans-4- (4- (2-((3-carbamoylphenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid Methyl;
Trans-4- (4- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid methyl;
Trans-4- (4- (2- (ethyl (3-methoxyphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid methyl;
Trans-4- (4- (2- (ethyl (o-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid methyl;
Trans-4- (4- (2- (6-methyl-3,4-dihydroquinolin-1 (2H) -yl) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide ) Methyl cyclohexane-1 -carboxylate;
Trans-4- (4- (2- (ethyl (p-tolyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid methyl;
Trans-4- (4- (2- (ethyl (4- (hydroxymethyl) phenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carvone Methyl acid;
Trans-4- (1- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -1H-indole-2-carboxamide) methyl cyclohexane-1-carboxylate;
Trans-4- (1- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -6-methoxy-1H-indole-2-carboxamido) cyclohexane-1-carboxylate methyl;
Trans-4- (4- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -4H-furo [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid methyl;
4- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -N- (trans-4-((6-hydroxyhexyl) carbamoyl) cyclohexyl) -4H-thieno [3,2-b] Pyrrole-5-carboxamide;
6- (trans-4- (4- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1-carboxamide ) Hexane-1-sulfonic acid sodium;
Trans-4- (4- (2- (ethyl (3-isopropylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid methyl;
Trans-4- (4- (2- (ethyl (3- (hydroxymethyl) phenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid Methyl acid;
Trans-4- (4- (2-((3- (bromomethyl) phenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1- Methyl carboxylate;
Trans-4- (4- (2-((3- (dimethylamino) phenyl) (ethyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamide) cyclohexane-1 -Methyl carboxylate;
Trans-4- (4- (2- (ethyl (3-isobutylphenyl) amino) -2-oxoethyl) -4H-thieno [3,2-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylic acid methyl;
Trans-4- (1- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -1H-indole-2-carboxamide) methyl cyclohexane-1-carboxylate;
1- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -N- (trans-4-((6-hydroxyhexyl) carbamoyl) cyclohexyl) -1H-indole-2-carboxamide;
6- (trans-4- (1- (2- (ethyl (3-ethylphenyl) amino) -2-oxoethyl) -1H-indole-2-carboxamido) cyclohexane-1-carboxamido) hexane-1-sulfonic acid sodium ;
Trans-4- (1- (2- (ethyl (m-tolyl) amino) -2-oxoethyl) -1H-pyrrole-2-carboxamide) methyl cyclohexane-1-carboxylate; The compound according to claim 1, which is selected from the group consisting of methyl 2- (ethyl (m-tolyl) amino) -2-oxoethyl) -6H-thieno [2,3-b] pyrrole-5-carboxamido) cyclohexane-1-carboxylate. The compound as described in 1. Contacting the Oplophorus-derived luciferase with the compound of any one of claims 1-36,
A method of inhibiting the Oplophorus-derived luciferase.   38. The method of claim 37, wherein the Oplophorus-derived luciferase comprises the polypeptide sequence of SEQ ID NO: 2. A method for inhibiting Oplophorus-derived luciferase, comprising
The Oplophorus-derived luciferase is a compound of the formula (II):
(II)
Including contacting with
During the ceremony:
The dotted lines indicate the presence or absence of a bond;
n is 0, 1, 2, 3, 4 or 5;
X is CH, N, O, or S;
When X represents CH or N when the dotted line represents the presence of a bond, X is O or S when the dotted line represents the absence of a bond;
A is an optionally substituted phenyl ring, or an optionally substituted 5 or 6 membered heteroaryl ring;
R ¹ and R ² are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally Selected from the group consisting of alkoxyalkyl substituted with and optionally substituted alkoxyalkoxyalkyl;
Z is selected from the group consisting of -NR ³ R ⁴ and -OR ⁵ ; and R ³ , R ⁴ and R ⁵ are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, Optionally substituted C ₃ -C ₈ -cycloalkyl, optionally substituted C ₃ -C ₈ -cycloalkylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted hetero Selected from the group consisting of aryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, and optionally substituted heterocyclylalkyl; or R ³ and R ⁴ with the nitrogen atom to which they are attached Together, optionally together forming a ring.   40. The method of claim 39, wherein the Oplophorus derived luciferase comprises the polypeptide sequence of SEQ ID NO: 2. A method of controlling luminescence of Oplophorus-derived luciferase in a sample, comprising
(A) contacting the sample with coelenterazine substrate and a compound according to any of claims 1 to 36; and (b) detecting luminescence in the sample,
Including
37. The method wherein the compound according to any one of claims 1-36 causes a decrease in luminescence from the Oplophorus-derived luciferase. A method of detecting an interaction between a first protein and a second protein in a sample comprising:
(A) contacting the sample with coelenterazine substrate and a compound according to any of claims 1 to 36, said sample being
(Xvii) a first polynucleotide encoding a first fusion protein comprising a first fragment of Oplophorus-derived luciferase and a first protein; and (xviii) a second fragment of the Oplophorus-derived luciferase and a second protein A second polynucleotide encoding a second fusion protein, and (b) detecting luminescence in said sample,
Including
The method wherein the detection of luminescence indicates an interaction between the first protein and the second protein.   43. A method according to any one of claims 41 to 42, comprising contacting the sample with the coelenterazine substrate prior to contacting the sample with a compound according to any one of claims 1-36. .   When the first protein and the second protein interact, the first fragment of the Oplophorus-derived luciferase and the second fragment of the Oplophorus-derived luciferase are stably bound to the coelenterazine substrate 43. The method of claim 42, wherein possible full-length enzymes are reconstituted. A method of detecting an interaction between a first protein and a second protein in a sample comprising:
(A) contacting the sample with coelenterazine substrate and a compound according to any one of claims 1 to 36, said sample being:
(Xix) a first polynucleotide encoding a first fusion protein comprising a bioluminescent donor Oplophorus-derived luciferase and a first protein; and (xx) a second fusion comprising a fluorescent acceptor molecule and a second protein Comprising a second polynucleotide encoding a protein;
(B) detecting bioluminescence resonance energy transfer (BRET) in the sample indicative of interaction or proximity of the bioluminescence donor and the fluorescence acceptor.   46. The method of any one of claims 41-45, wherein the sample comprises cells.   47. The method of claim 46, wherein the cells comprise the Oplophorus derived luciferase.   47. The method of claim 46, wherein the cells express the Oplophorus derived luciferase.   49. The method according to any one of claims 41 to 48, wherein the coelenterazine substrate is coelenterazine, coelenterazine derivative, coelenterazine analogue, procoelenterazine, or quinone masked coelenterazine.   A first fusion protein comprising a first target protein and a bioluminescent donor molecule, wherein said bioluminescent donor molecule is an Oplophorus-derived luciferase; a second fusion protein comprising a second target protein and a fluorescent acceptor molecule; A bioluminescent resonance energy transfer (BRET) system comprising a coelenterazine substrate, and a compound according to any of claims 1-36. A kit comprising: (a) the compound according to any one of claims 1-36; and (b) an Oplophorus-derived luciferase.   52. The kit of claim 51, wherein the Oplophorus derived luciferase comprises the polypeptide sequence of SEQ ID NO: 2.   53. The kit of any one of claims 51-52, further comprising a coelenterazine substrate.   54. The kit of any one of claims 51-53, further comprising instructions for performing a luminescence assay. A method of controlling luminescence of Oplophorus-derived luciferase in a sample, comprising
(A) contacting the sample with coelenterazine substrate and a compound of formula (II):
(II)
Including
During the ceremony:
The dotted lines indicate the presence or absence of a bond;
n is 0, 1, 2, 3, 4 or 5;
X is CH, N, O, or S;
When X represents CH or N when the dotted line represents the presence of a bond, X is O or S when the dotted line represents the absence of a bond;
A is an optionally substituted phenyl ring, or an optionally substituted 5 or 6 membered heteroaryl ring;
R ¹ and R ² are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally Selected from the group consisting of alkoxyalkyl substituted with and optionally substituted alkoxyalkoxyalkyl;
Z is selected from the group consisting of -NR ³ R ⁴ and -OR ⁵ ; and R ³ , R ⁴ and R ⁵ are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, Optionally substituted C ₃ -C ₈ -cycloalkyl, optionally substituted C ₃ -C ₈ -cycloalkylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted hetero Selected from the group consisting of aryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, and optionally substituted heterocyclylalkyl; or R ³ and R ⁴ with the nitrogen atom to which they are attached Together forming an optionally substituted ring together; and (b) detecting luminescence in said sample,
The method wherein the compound of formula (II) causes a decrease in luminescence from the Oplophorus-derived luciferase. A method of detecting an interaction between a first protein and a second protein in a sample comprising:
(A) contacting the sample with coelenterazine substrate and a compound of formula (II),
(II)
During the ceremony:
The dotted lines indicate the presence or absence of a bond;
n is 0, 1, 2, 3, 4 or 5;
X is CH, N, O, or S;
When X represents CH or N when the dotted line represents the presence of a bond, X is O or S when the dotted line represents the absence of a bond;
A is an optionally substituted phenyl ring, or an optionally substituted 5 or 6 membered heteroaryl ring;
R ¹ and R ² are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally Selected from the group consisting of alkoxyalkyl substituted with and optionally substituted alkoxyalkoxyalkyl;
Z is selected from the group consisting of -NR ³ R ⁴ and -OR ⁵ ; and R ³ , R ⁴ and R ⁵ are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, Optionally substituted C ₃ -C ₈ -cycloalkyl, optionally substituted C ₃ -C ₈ -cycloalkylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted hetero Selected from the group consisting of aryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, and optionally substituted heterocyclylalkyl; or R ³ and R ⁴ with the nitrogen atom to which they are attached Together, form an optionally substituted ring together,
The sample is
(Xxi) a first polynucleotide encoding a first fusion protein comprising a first fragment of Oplophorus-derived luciferase and a first protein; and (xxii) a second fragment of the Oplophorus-derived luciferase and a second protein A second polynucleotide encoding a second fusion protein comprising: and (b) detecting luminescence in said sample,
The method wherein the detection of luminescence indicates an interaction between the first protein and the second protein.   57. The method of any one of claims 55-56, comprising contacting the sample with the coelenterazine substrate prior to contacting the sample with the compound of formula (II).   When the first protein and the second protein interact, the first fragment of the Oplophorus-derived luciferase and the second fragment of the Oplophorus-derived luciferase are stably bound to the coelenterazine substrate 58. The method of claim 57, wherein possible full length enzymes are reconstituted. A method of detecting an interaction between a first protein and a second protein in a sample comprising:
(A) contacting the sample with coelenterazine substrate and a compound of formula (II),
(II)
During the ceremony:
The dotted lines indicate the presence or absence of a bond;
n is 0, 1, 2, 3, 4 or 5;
X is CH, N, O, or S;
When X represents CH or N when the dotted line represents the presence of a bond, X is O or S when the dotted line represents the absence of a bond;
A is an optionally substituted phenyl ring, or an optionally substituted 5 or 6 membered heteroaryl ring;
R ¹ and R ² are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally Selected from the group consisting of alkoxyalkyl substituted with and optionally substituted alkoxyalkoxyalkyl;
Z is selected from the group consisting of -NR ³ R ⁴ and -OR ⁵ ; and R ³ , R ⁴ and R ⁵ are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, Optionally substituted C ₃ -C ₈ -cycloalkyl, optionally substituted C ₃ -C ₈ -cycloalkylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted hetero Selected from the group consisting of aryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, and optionally substituted heterocyclylalkyl; or R ³ and R ⁴ with the nitrogen atom to which they are attached Together, form an optionally substituted ring together,
The sample is
(Xxiii) a first polynucleotide encoding a first fusion protein comprising a bioluminescent donor Oplophorus-derived luciferase and a first protein; and (xxiv) a second fusion comprising a fluorescent acceptor molecule and a second protein Comprising a second polynucleotide encoding a protein;
(B) detecting bioluminescence resonance energy transfer (BRET) in the sample indicative of interaction or proximity of the bioluminescence donor and the fluorescence acceptor.   60. The method of any one of claims 55-59, wherein the sample comprises cells.   61. The method of claim 60, wherein the cells comprise the Oplophorus derived luciferase.   61. The method of claim 60, wherein the cells express the Oplophorus derived luciferase.   63. The method of any one of claims 55-62, wherein the coelenterazine substrate is coelenterazine, coelenterazine derivative, coelenterazine analog, procoelenterazine, or quinone masked coelenterazine. A bioluminescent resonance energy transfer (BRET) system,
A first fusion protein comprising a first target protein and a bioluminescent donor molecule, wherein said bioluminescent donor molecule is an Oplophorus-derived luciferase; a second fusion protein comprising a second target protein and a fluorescent acceptor molecule; A coelenterazine substrate, and a compound of formula (II),
(II)
During the ceremony:
The dotted lines indicate the presence or absence of a bond;
n is 0, 1, 2, 3, 4 or 5;
X is CH, N, O, or S;
When X represents CH or N when the dotted line represents the presence of a bond, X is O or S when the dotted line represents the absence of a bond;
A is an optionally substituted phenyl ring, or an optionally substituted 5 or 6 membered heteroaryl ring;
R ¹ and R ² are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally Selected from the group consisting of alkoxyalkyl substituted with and optionally substituted alkoxyalkoxyalkyl;
Z is selected from the group consisting of -NR ³ R ⁴ and -OR ⁵ ; and R ³ , R ⁴ and R ⁵ are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, Optionally substituted C ₃ -C ₈ -cycloalkyl, optionally substituted C ₃ -C ₈ -cycloalkylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted hetero Selected from the group consisting of aryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, and optionally substituted heterocyclylalkyl; or R ³ and R ⁴ with the nitrogen atom to which they are attached Said bioluminescent resonance energy transfer (BRET) system, together forming an optionally substituted ring. (A) Compound of Formula (II):
(II)
During the ceremony:
The dotted lines indicate the presence or absence of a bond;
n is 0, 1, 2, 3, 4 or 5;
X is CH, N, O, or S;
When X represents CH or N when the dotted line represents the presence of a bond, X is O or S when the dotted line represents the absence of a bond;
A is an optionally substituted phenyl ring, or an optionally substituted 5 or 6 membered heteroaryl ring;
R ¹ and R ² are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally Selected from the group consisting of alkoxyalkyl substituted with and optionally substituted alkoxyalkoxyalkyl;
Z is selected from the group consisting of -NR ³ R ⁴ and -OR ⁵ ; and R ³ , R ⁴ and R ⁵ are each independently hydrogen, optionally substituted C ₁ -C ₈ alkyl, Optionally substituted C ₃ -C ₈ -cycloalkyl, optionally substituted C ₃ -C ₈ -cycloalkylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted hetero Selected from the group consisting of aryl, optionally substituted heteroarylalkyl, optionally substituted heterocyclyl, and optionally substituted heterocyclylalkyl; or R ³ and R ⁴ with the nitrogen atom to which they are attached Together, together form an optionally substituted ring; and (b) a luciferase comprising Oplophorus.   66. The kit of claim 65, wherein the Oplophorus derived luciferase comprises the polypeptide sequence of SEQ ID NO: 2.   67. The kit of any one of claims 65-66, further comprising a coelenterazine substrate.   68. The kit of any one of claims 65-67, further comprising instructions for performing a luminescence assay.